Telomeres and Telomerase

Part 1: The Roles of Telomeres and Telomerase

00:00:03.20 Hello, my name's Elizabeth Blackburn. I'm in the 00:00:06.25 Department of Biochemistry and Biophysics at the 00:00:09.19 University of California, San Francisco. And in this set of 00:00:13.19 lectures, I'm going to talk about telomeres and 00:00:16.08 telomerase. And I'll get to their implications for human 00:00:20.19 health and disease. The first part of this series of three 00:00:26.07 lectures is going to introduce you to the roles of telomeres 00:00:30.18 and telomerase. So, let's begin by focusing in on what 00:00:36.27 goes on at the very heart of a cell. Now if you looked at a 00:00:42.10 cell which is just about to divide, you looked into the 00:00:46.06 microscope and you stained the chromosomes, this is 00:00:48.14 what you would see. Those blue, double sausage-like 00:00:51.25 objects that you can see all over this microscope slide are 00:00:57.17 the human chromosomes, and the DNA has just 00:00:59.24 duplicated, which is why they look double. Now if you 00:01:03.11 look closely, you can see red spots, two red spots at the 00:01:06.15 ends of every double chromosome pair. And these red 00:01:11.15 spots are a molecular probe that's lighting up the telomeric 00:01:15.05 DNA that's found in common at all of the chromosome 00:01:18.25 ends. And I'm going to tell you a lot about that telomeric 00:01:23.04 DNA. So why are telomeres important? Their role is to cap 00:01:30.14 off the ends of chromosomes. So that's a simple concept, 00:01:35.26 but we can dive down into it further and think a little bit 00:01:38.17 more about what that actually means. So when we think 00:01:42.07 of the end of the DNA, there can be two kinds: There 00:01:45.09 can be the natural end of the DNA, such as the ones we 00:01:48.07 see here, the ends of the chromosomal DNA; and there 00:01:52.12 can also be DNA breaks. Now, the job of a cell is to seal 00:02:01.05 up any DNA breaks that happen by accident. And so a 00:02:05.03 major part of this capping function, which is one of the 00:02:09.04 aspects of telomere functions, is to prevent the telomeres 00:02:13.23 from undergoing those very DNA transactions of very 00:02:17.11 DNA reactions that are undergone by a broken DNA end. 00:02:24.00 So if you have a break in the DNA, it can be sutured 00:02:27.14 together by, for example, recombination or just simply the 00:02:31.21 two broken ends can be ligated right back together by 00:02:34.25 end-to-end fusions. Also, such DNA breaks are subject to 00:02:40.15 degradation. Now the telomere protects, it caps, the end 00:02:45.05 of the chromosome and protects against all of these kinds 00:02:48.13 of things that would normally happen to a broken DNA 00:02:52.12 end. How does it do that? The DNA sequences that you 00:02:58.08 find at the ends of chromosomes, repeated over and 00:03:01.15 over, are fairly similar in nature to each other. They're 00:03:06.11 relatively simple sequences, and they're too simple to 00:03:09.24 code for any proteins. They're not genes in the sense of 00:03:15.09 coding for any proteins or RNAs. In humans for example, 00:03:21.20 this repeated sequence is found up to a few thousand 00:03:24.14 times, tandemly repeated over and over at the ends of the 00:03:29.08 chromosomes. Another feature of the chromosomal DNAs 00:03:34.06 is that, of course, unlike most of the DNA of a 00:03:38.00 chromosome, which is duplex DNA, double-stranded 00:03:40.11 DNA, the very end is single-stranded. And in fact the 00:03:44.26 DNA strand is oriented 5' to 3', going toward the end of 00:03:50.03 the chromosome. And that turns out to be important. So, 00:03:58.12 we have now the telomere structure to begin with: We 00:04:01.27 have again, if we blow up the end of a chromosome, we 00:04:05.23 have these highly repeated sequences made up of a G- 00:04:10.12 rich sequence that's the repeat unit repeated over and 00:04:13.13 over again. As I said, it doesn't encode any protein 00:04:17.13 sequences, but each of these repeated sequences is like 00:04:23.05 a little attractive magnet for specific proteins that bind 00:04:27.25 sequence specifically to the telomeric DNA. They bind to 00:04:31.22 the telomeric repeats for the double-stranded portion, and 00:04:35.27 some of them bind the single-stranded portion, and that's 00:04:39.06 actually the G-rich strand that's forming the overhanging 00:04:45.11 end here. These together make some form of higher order 00:04:53.02 architecture we don't understand. We understand a lot 00:04:56.13 about the protein-DNA interactions, some of the molecular 00:04:59.11 details of that. Some of the details of the protein-protein 00:05:02.19 interactions in this complex, but we don't really 00:05:05.07 understand the higher order structure, so that's still a 00:05:07.22 challenge in the field. So I've just shown you a functional 00:05:14.04 telomere, but if telomeres cease to function... and we use 00:05:18.10 the term "telomere dysfunction" to just describe that 00:05:21.21 general state of the telomere that is not carrying out those 00:05:25.04 capping functions and other functions that I will get to... 00:05:30.07 there are a couple of different ways this can happen. The 00:05:33.09 first is if the tract of telomeric repeat is simply too short, 00:05:38.27 there's just not enough of the length of the repeats to form 00:05:44.27 a nice, long array that can form this higher order structure 00:05:48.27 that's necessary. This kind of dysfunction caused by the 00:05:54.27 shortening of the telomere, that can happen naturally, and 00:05:59.28 it does, and we'll get back to that in a moment, because 00:06:02.24 this is going to be an important part of these lectures, and 00:06:05.28 in fact it'll be really the focus of the third lecture in this 00:06:10.00 series. The other way that telomeres can become 00:06:15.07 dysfunctional: If for one reason or another, experimentally 00:06:19.12 induced, most commonly, one of these proteins cannot 00:06:23.27 bind correctly to the telomeric DNA; if its binding is 00:06:27.19 disrupted through some molecular intervention or other... 00:06:32.22 in both cases, cells sense and respond to this state of 00:06:40.03 telomere dysfunction. Now indeed, cells have a lot of very 00:06:46.22 strict regulatory reactions to the lack of proper telomeric 00:06:53.03 DNA. And the consequences for the cell is that, usually 00:06:59.06 this state of the cell's telomere dysfunction, through one 00:07:05.02 reason or another, will mean that the cell will cease to 00:07:10.20 divide. So this limits cell renewal capability if this happens 00:07:15.16 to one or more of its telomeres in the cell. If by chance the 00:07:20.20 cell does continue to multiply, now those telomeres 00:07:26.21 become subject to the very kinds of fusions (the DNA- 00:07:31.28 joining events that I told you telomeres shouldn't allow to 00:07:35.11 happen), and that can lead to genomic instability, 00:07:39.21 because the end-to-end joining of telomeres to 00:07:42.17 themselves (other telomeres, that is) or to broken DNAs, 00:07:47.00 that can cause the chromosomes, which fuse to each 00:07:52.07 other, to tear themselves apart as the cells divide, leading 00:07:56.09 to genomic instability. So clearly telomere function is very 00:08:02.20 important for cells. And in fact, one of the consequences 00:08:07.10 of genomic instability in human cells is that the cells can 00:08:11.13 become cancerous. I'm just going to show you a picture. 00:08:17.26 If you look under a microscope of some cells in which 00:08:22.20 we've disrupted one of the telomeric proteins, what you 00:08:25.22 can see is... remember I told you the blue, double things 00:08:30.01 are the chromosomal DNAs... and look, here's a 00:08:32.29 chromosome here in which there's been a telomere 00:08:35.19 fusion. So here's the two telomeres at the end, here's the 00:08:38.25 other two all the way here, but there are fused telomeres 00:08:43.27 here. So this is now a chromosome that has two 00:08:47.15 centromeres, it's got a centromere here, it's a got a 00:08:50.02 centromere here, and if those two centromeres try to pull 00:08:54.05 apart in a cell that is dividing, the chromosome will get 00:08:59.15 ripped apart. Here's another example of such an end-to- 00:09:04.22 end fused chromosome. This kind of change can happen 00:09:09.05 if you disrupt the telomeric integrity by, for example, 00:09:13.05 disrupting the binding of proteins. The other kind of 00:09:18.23 function, it's related, but we can distinguish it, the other 00:09:23.02 kind of function of telomeres is that they have to allow for 00:09:27.13 the complete replication of the telomeric DNA. So what's 00:09:33.07 the issue here? Well, the mechanism of DNA replication, 00:09:40.12 the machinery of DNA replication, has a particular quirk to 00:09:44.23 it. It's very good at faithfully copying almost all the way 00:09:49.19 along the length of the chromosomal DNA (or any linear 00:09:53.17 DNA), but the make-up of the DNA replication machinery 00:09:59.00 is such that is cannot copy the very, very end of the linear 00:10:04.28 DNA, such as a eukaryotic chromosomal DNA. Now the 00:10:10.11 predicted and observed consequence of that inability is 00:10:14.22 that, each time the DNA replicates, which is has to do as 00:10:20.01 the cell divides, and then the cell divides, the daughter 00:10:24.00 DNAs are predicted to become shorter and shorter and 00:10:27.13 shorter. And this is just a simple consequence of the 00:10:32.15 nature of the DNA replication machinery that is otherwise 00:10:37.00 so good at replicating all the way along all the length of 00:10:41.07 the chromosomal DNA. But the very ends cannot be 00:10:45.09 completely replicated without some form of compensatory 00:10:49.20 mechanism. So what's the consequence of this loss? 00:10:53.20 Well, obviously, something has to compensate in the long 00:10:56.06 run, otherwise, we wouldn't be here. But even on a 00:10:59.29 shorter timeframe, as cells divide and divide, the DNA 00:11:03.08 gets shorter and shorter, one might predict that there 00:11:05.24 would come a point when there wouldn't be enough of 00:11:10.10 something or other at the end that then the chromosomes 00:11:14.19 would no longer be able to support cell division, and the 00:11:18.00 cells might eventually undergo what's called senescence. 00:11:22.00 And indeed James Watson in 1972, just considering the 00:11:26.02 mechanism of DNA replication, proposed this constant 00:11:30.15 shortening problem, and Olovnikov around the same time 00:11:36.20 proposed that in fact perhaps such loss of terminal DNA, 00:11:41.22 without knowing at that stage what the molecular nature 00:11:44.07 of the terminal DNA was, Olovnikov proposed that 00:11:47.12 perhaps such gradual loss could be something that 00:11:50.20 underlies the eventual senescence of cells that is seen 00:11:54.14 sometimes when, for example, human cells are grown in 00:11:57.08 culture. This was a prescient idea because, in fact, this 00:12:02.04 indeed has been found to be one of the causes of why 00:12:07.17 human cells cannot replicate themselves, the cells cannot 00:12:12.18 proliferate, indefinitely in culture. So, shortening of 00:12:19.26 telomeric DNA is something that will be problematic for 00:12:25.12 cells. How is this problem solved? Well, it's solved by an 00:12:33.01 enzyme called telomerase. So I'd like to introduce you to 00:12:37.12 telomerase now, and how it was found. Telomerase was 00:12:46.06 sought because there was a set of accumulating 00:12:49.24 observations on telomeric DNA in cells, the telomeric 00:12:54.24 DNA as it was in cells in vivo, that couldn't be readily 00:12:58.22 explained by what was currently known about DNA 00:13:02.12 replication or DNA recombination or other kinds of DNA 00:13:06.22 reactions at the time, which was the late 1970s, early 00:13:10.21 1980s. And let me give you some examples of such 00:13:15.14 puzzling observations. Well, the first one was that in the 00:13:21.26 ciliated protozoan Tetrahymena, which has a lot of very 00:13:24.26 small mini-chromosomes and is therefore amenable to 00:13:28.02 molecular analyses of its telomeric DNA by direct 00:13:31.09 methods, the telomeric repeat sequences (remember I told 00:13:36.05 you about the repeated sequences repeated over and 00:13:38.16 over at the ends of chromosomes)... the repeat sequence 00:13:41.28 in this organism, G4T2 repeats, were heterogeneous in 00:13:48.05 their number, in different molecules in a population of 00:13:53.01 otherwise homogeneous cells. And heterogeneous in this 00:13:57.20 setting here is meaning, these were different in number, 00:14:04.04 so some copies of the mini-chromosome would have 00:14:07.07 twenty repeats on the end, some would have 50-some, 00:14:09.09 49-some, 82-some, 53... they all had different numbers of 00:14:13.16 repeats in this sort of more-or-less normal distribution. So 00:14:18.18 that was very surprising, because if you look across at the 00:14:21.15 internal region of a DNA, such as a chromosomal DNA, if 00:14:25.20 you look at one cell compared with another in a 00:14:29.05 population of cells from, say, one organism, it should 00:14:32.08 always be an identical sequence. So here were different 00:14:35.21 numbers of repeats at the ends of different molecules in a 00:14:40.15 population of cells that should have otherwise been 00:14:43.07 homogeneous, and were homogeneous in their internal 00:14:46.09 regions of the chromosome. Now, a second kind of 00:14:50.17 observation was a somewhat more complicated one, and 00:14:55.14 it means that I have to just take a moment to tell you 00:14:59.12 about the lifecycle of a particular group of ciliated 00:15:02.23 protozoans, which the species Tetrahymena in which 00:15:07.08 these G4T2 repeat tracts were found to be the telomeric 00:15:10.26 tracts. The Tetrahymena cells go through a lifecycle 00:15:15.24 stage where they have a somatic nucleus which 00:15:20.09 undergoes developmentally controlled fragmentation. And 00:15:24.16 fascinatingly, telomeric DNA sequences were added 00:15:27.29 directly to those freshly formed DNA ends, making from 00:15:33.27 longer chromosomes a series of shorter mini-chromosomes. 00:15:38.25 How did that telomeric DNA get added to 00:15:41.15 the ends? It wasn't clear. The third observation came 00:15:48.11 from observing the cells of an organism which causes 00:15:52.09 sleeping sickness, a single-celled parasitic organism 00:15:55.24 called a trypanosome. And these were being propagated 00:15:59.07 in the laboratory setting, and what was found was that the 00:16:04.05 telomeric DNA restriction fragments, the end fragments of 00:16:08.05 the chromosomes in these organisms, were gradually 00:16:11.09 getting longer and longer and longer. And this didn't look 00:16:14.10 like, say, recombination, and certainly was not expected 00:16:18.01 for normal, as one thought about it, DNA replication. The 00:16:23.27 fourth observation was again something that came out an 00:16:29.00 experiment that I'll have to explain. Now one could put 00:16:33.07 circular plasmids into yeast cells, and those plasmids are 00:16:37.01 linearized, essentially they're unstable, they get gobbled 00:16:40.16 up or, rarely, will recombine into the chromosomes and 00:16:44.12 thus be preserved. But what was found was that if one 00:16:48.10 simply grafted onto the ends of such a linearized and 00:16:52.24 therefore normally very unstable yeast plasmid, if one 00:16:56.24 grafted onto its ends Tetrahymena telomeric DNA 00:17:01.15 fragments (the telomeres of Tetrahymena mini- 00:17:03.24 chromosomes) and introduced those into cells... so the 00:17:08.01 grafting was done in vitro with enzymes and the purified 00:17:11.09 DNA molecules, and then the resulting hybrid of the yeast 00:17:17.05 linearized plasmid and the telomeres added onto its end, 00:17:21.29 that was introduced into the yeast cells, those were 00:17:25.00 maintained in yeast cells as linear mini-chromosomes, and 00:17:28.12 yeast telomeric DNA repeat were grafted on somehow 00:17:34.11 inside the yeast cells, to the ends of the Tetrahymena 00:17:39.02 telomeric repeats. And that didn't look like a reaction one 00:17:43.11 would expect from standard known models of DNA 00:17:46.26 replication or recombination. All of these suggested that 00:17:52.03 perhaps there was some capability of cells to add 00:17:57.16 telomeres. And that idea, in a conceptual way, was given 00:18:04.22 some force by an observation made by the noted 00:18:09.25 geneticist, Barbara McClintock, who worked with maize 00:18:13.06 (corn), and she noted that a particular maize mutant stock 00:18:17.28 lost the capacity to do something that is normally found in 00:18:23.24 normal, wild-type maize. In such wild-type maize, if a 00:18:28.28 chromosome is broken by, for example, exo-radiation or 00:18:32.15 some mechanical rupture at a particular stage in 00:18:37.07 development, then that broken end can be, as she 00:18:40.18 described it, "healed." It becomes a normal, stable 00:18:43.29 telomere. Nobody knew the molecular basis of that. And 00:18:47.29 she found a mutant that had lost that capacity. And when 00:18:51.04 you see a mutant where something is changed, you have 00:18:53.17 a feeling that that's reflective of a cellular process that 00:18:57.21 can take place, and that it's not just by chance that this 00:19:01.08 healing event was taking place. All of which, then, 00:19:07.23 focused in on the question of: Was there a new enzyme 00:19:10.02 that worked in cells that could extend telomeric DNA? So 00:19:14.17 we sought such an enzyme in the early to mid-1980s. And 00:19:19.24 we used for that purpose the single-celled, ciliated 00:19:24.18 protozoan Tetrahymena thermophila, shown in this 00:19:27.18 scanning electron micrograph here. And you can see the 00:19:31.02 cilia are on the cell surface. This experimental system was 00:19:34.26 chosen because the organism contains large numbers of 00:19:40.00 very short mini-chromosome, therefore, large numbers of 00:19:44.15 telomeres, and therefore, one would reason, perhaps if 00:19:49.03 there were such an enzyme that existed that could add 00:19:53.03 telomeric DNA to the end of chromosomes or to 00:19:56.25 preexisting telomeres, then this would be a potentially 00:20:00.05 good source for it because there's a lot of telomeres in 00:20:03.28 this organism. And indeed that proved to be the case. 00:20:08.18 And Carol Greider, my then-graduate student, joined the 00:20:12.11 lab in 1984, and here's a picture of Carol, freshly from 00:20:16.10 visiting Southern California, and looking at an 00:20:20.29 autoradiogram shown with this x-ray film here in the lab. 00:20:26.17 And we together found this enzyme telomerase. Now let 00:20:32.15 me show you briefly what we did. We took a mimic of that 00:20:37.18 overhanging G-rich strand DNA that's normally found at 00:20:41.11 the ends of chromosomes, in the form of an oligonucleotide, 00:20:45.18 and here it is shown here. And I've just 00:20:47.13 colored the bases different colors so that we can see 00:20:50.22 them easily. And here's its 3' hydroxyl end. We mixed it 00:20:55.28 with an extract of Tetrahymena cells, and this worked 00:20:59.13 particularly well at the stage in development that I 00:21:03.22 mentioned to you earlier... that stage at which telomeric 00:21:07.20 DNA is added to freshly broken ends of chromosomes. 00:21:12.09 We reasoned that that would be a particularly good time 00:21:15.29 to find such an activity, thinking it might be likely to be 00:21:20.16 induced or present in perhaps larger-than-normal 00:21:23.25 amounts, because this would be a time when there'd be a 00:21:27.02 demand for such an enzyme. All of this was hypothetical 00:21:30.00 at the time, but by putting in an appropriate mix of things 00:21:33.17 that are often added to make polymerases happy (such 00:21:39.02 as magnesium ion), and just by using two nucleoside 00:21:43.17 triphosphate precursors, dGTP and TTP, we were able to 00:21:48.21 find that the telomeric DNA indeed was added to the end 00:21:53.02 of such an oligonucleotide. In fact, in the test tube, a 00:21:56.09 large number of repeats could be added. So we get a lot 00:22:00.22 of repeats added, through eventually something limits the 00:22:06.18 addition. So this is what telomerase did. Now how is it 00:22:14.16 doing that? It was adding a given sequence. The first 00:22:18.09 clue came from the observation... and here I'm just 00:22:22.01 showing you a nice picture of the products run on a DNA 00:22:27.10 sequencing gel, just to give you feel for what it looks like. 00:22:30.14 We'll just look at one of these groups, so here are 00:22:33.13 different time points, increasing number of minutes after 00:22:37.09 incubating this input primer with dGTP and TTP. The 00:22:41.06 dGTP was radiolabeled, so we're just looking at an 00:22:43.26 autoradiogram. Every time we see a labeled band, this is 00:22:47.22 a product that's been added, so the input would run here. 00:22:50.20 If you added one, two, three, four nucleotides, you could 00:22:53.11 see you'd get longer and longer bands. And what you can 00:22:56.20 see is that there is kind of a striped pattern. Every six 00:23:01.05 nucleotides, there was a pause in the addition, and so 00:23:04.16 you could see a pattern of six nucleotides being added, 00:23:09.03 and you could see more and more repeats, as shown by 00:23:11.20 the bands getting higher and higher and higher, were 00:23:14.18 being added with time. Now, certain clues came. Not any 00:23:21.27 nucleotide could work. So, two examples of the telomeric 00:23:27.01 DNA, where we've got a permutation ending with four Gs 00:23:30.12 here, or a permutation ending with two Ts here. These 00:23:34.26 were each competent for addition by this activity. 00:23:41.04 However, the complementary strand, and again, I've just 00:23:44.07 colored the bases distinctively and given you two 00:23:47.01 examples... the complementary strand oligonucleotides 00:23:50.25 were not competent for such addition. Another interesting 00:23:59.04 clue came when we looked in more detail at different 00:24:02.16 oligonucleotides that could act as primers. Now, we had 00:24:10.01 found, as I said to you, that when you put Tetrahymena 00:24:13.20 telomeric repeats into yeast cells, live cells, then yeast 00:24:18.12 repeats were added somehow, it was unknown then, to 00:24:22.15 the Tetrahymena telomeres. And the yeast repeats have 00:24:27.06 a different sequence, and I've shown you a little segment 00:24:29.06 of it. It's T, G, sometimes one G, sometimes two, 00:24:34.13 sometimes three Gs. So we wondered if the converse 00:24:38.23 would be the case; if we added a yeast primer to a 00:24:43.14 Tetrahymena extract, now in vitro, would we see 00:24:48.04 Tetrahymena sequences added to the yeast telomeric 00:24:52.19 oligonucleotide. And indeed, we did see such addition. 00:24:58.26 And again, large numbers of repeats could be added to 00:25:02.14 such a primer quite efficiently. Now we also noticed 00:25:08.19 another interesting thing about the reaction. When we 00:25:12.21 had a primer that ended with four Gs, what we found was 00:25:17.15 that first two Ts were added, and then four Gs, two Ts, 00:25:20.15 and so on. When we had a primer that ended with three 00:25:23.06 Gs, first a G was added, and then the two Ts, in other 00:25:27.19 words completing this run of four Gs. And I haven't shown 00:25:30.26 it, but if you had a primer that ended in two Gs, then GG, 00:25:35.07 and then TT. So this suggested that perhaps something 00:25:39.22 was aligning this very first portion, the portion of the 00:25:46.29 reaction that's taking place, adding to the very 3' end of 00:25:50.09 the primer, that perhaps something was aligning this, in 00:25:53.29 the enzyme somehow. Because in other words, if you 00:25:58.10 lined it up like this, then everything was lined up. What 00:26:01.08 could be doing this? So was there something that aligned 00:26:04.28 the product-forming part of this enzyme? Remember this 00:26:10.17 was very mysterious at the time. So in fact we looked and 00:26:15.07 found that, indeed, as I said, you had four Gs, and you 00:26:19.25 added two Ts. If you had three Gs, one G was added. 00:26:23.08 Two Gs, and you'd have two Gs added, and then the two 00:26:25.14 Ts. And if you had one G, then it would be three Gs, and 00:26:29.08 then the two Ts. So in fact, there was something that was 00:26:31.27 aligning how the next repeats were added, dependent on 00:26:36.10 the 3' end of the primer. The result of all of this kind of 00:26:44.02 analysis led us to the idea that there was in fact a 00:26:47.15 template within the telomerase, and to our surprise, this 00:26:52.07 template turned out to be made of RNA, an RNA that is 00:26:56.25 actually built in to the telomerase complex. And this 00:27:01.04 template is a short portion of this RNA, which otherwise 00:27:05.26 has a lot of other structure which is built into the enzyme 00:27:09.20 particle, which contains also a protein, which is called 00:27:15.18 TERT. So what telomerase does is it takes that single- 00:27:20.18 stranded G-rich strand, it aligns the 3' end nucleotides by 00:27:27.07 Watson-Crick base pairing onto the template sequence, 00:27:31.29 so here's the example where we have two Gs at the end 00:27:35.05 of the primer. It aligns it on this part of the sequence, and 00:27:39.20 then it polymerizes, one at a time, each of these 00:27:44.29 nucleotides onto the DNA end, extending the DNA end 00:27:53.02 by copying this template. And so now, you end up with 00:27:58.23 longer DNA. So telomerase is a unique polymerase, it's a 00:28:06.22 reverse transcriptase by the definition, that is, copies RNA 00:28:12.18 into DNA. It's unique because the RNA component is 00:28:20.07 actually intrinsically built into the telomerase ribonucleoprotein 00:28:24.13 particle. It has to be built in for that 00:28:29.13 templating to take place. And indeed the enzyme is truly a 00:28:34.07 ribonucleoprotein enzyme, we believe. And unlike, for 00:28:39.26 example, the reverse transcriptases that copy, say, the 00:28:43.03 HIV viral genome, which is a genome thousands of 00:28:47.27 nucleotides long, a very complicated sequence which 00:28:51.14 encodes proteins, this enzyme copies very short 00:28:56.25 sequences over and over again. And it does it by this 00:29:00.26 process of aligning the DNA end on the template, and so 00:29:08.19 just to complete the thought here, here's the template. If 00:29:11.28 we polymerize all these together onto the DNA end, now 00:29:15.14 you'll have a new DNA end that ends with TTG, and that 00:29:19.26 will realign in the next round back in this AAC, for another 00:29:24.12 round of synthesis, and this can go on in the test tube for 00:29:28.07 many repeats. I've told you about some of the enzymatic 00:29:32.13 properties of telomerase, but what good is telomerase for 00:29:37.03 cells? Well, the answer came by manipulating telomerase 00:29:42.25 in Tetrahymena, the organism in which it was first 00:29:46.14 discovered. So if you remember, telomerase is adding 00:29:52.25 telomeric DNA to the ends of chromosomes, and so the 00:29:59.19 question was: What happened if it couldn't do that? So 00:30:03.17 we looked in Tetrahymena, which, as I said, was a good 00:30:07.03 source of telomerase, and another feature of these 00:30:10.23 organisms that I didn't tell you was that, if you grow them 00:30:14.00 in culture in the laboratory, they're effectively immortal, so 00:30:17.15 long as you keep them fed and under good conditions, 00:30:20.12 they can just propagate and propagate and propagate, 00:30:22.27 seemingly forever. And they have plenty of telomerase. 00:30:29.19 We manipulated the telomerase RNA. Now, we made 00:30:33.14 some changes in the RNA, and I won't go into the details 00:30:36.20 of it, but the effect of such small changes in the RNA I've 00:30:42.17 shown diagrammatically here. The telomeric DNA repeat 00:30:46.25 sequences, which as I said consist of multiple repeats at 00:30:51.17 the ends of chromosomes normally, as the cells went 00:30:56.17 through cell divisions, the telomeres progressively got 00:30:59.11 shorter and shorter, and after about 20 or 25 cell 00:31:02.20 divisions, the cells ceased to divide. So in other words, 00:31:07.07 when we inactivated telomerase, over the succeeding 00:31:11.16 cell divisions the telomeres progressively shortened, so in 00:31:15.18 effect when the cells ceased dividing, they'd become 00:31:19.07 mortal. From being immortal, they'd become mortal. And all 00:31:23.22 we had done was to inactivate telomerase by this small 00:31:28.23 change, so like a little stiletto stuck at the heart of 00:31:31.25 telomerase, we killed the enzyme very surgically, and we 00:31:35.08 were able to make immortal cells become mortal. So, the 00:31:40.17 conclusion is that the telomerase maintains the ends of 00:31:43.24 chromosomes. Telomeres are replenished by telomerase 00:31:47.07 as they keep dividing. And in fact, that continuing 00:31:51.22 replenishment, even in the face of the continuing 00:31:55.01 shortening processes that take place, can compensate 00:31:58.20 for those shortening processes and allow the cells to 00:32:01.15 keep on dividing. So that's what telomerase does for 00:32:05.28 cells. And that's the important message: Telomeres are 00:32:10.18 replenished by telomerase. Now, let's go into a more 00:32:16.03 detailed experiment in yeast cells. If we look at a culture 00:32:20.07 of yeast cells and we plate them out on a plate in a 00:32:24.03 laboratory... all those little white spots are cells that have 00:32:28.04 grown up from individual cells distributed on the plate, 00:32:30.24 they've been distributed on kind of a V-shape pattern 00:32:32.26 here, a triangular pattern. So, the cells are growing well 00:32:37.11 normally. Now if we delete one of the genes for 00:32:40.16 telomerase, either the RNA structural gene or the protein 00:32:43.26 structural gene for the core of the enzyme, now just as I 00:32:48.20 described for you in Tetrahymena, the cells divide and 00:32:54.16 divide, and the telomeres progressively get shorter and 00:32:57.03 shorter, until they get to a point where most of the cells 00:33:00.03 completely cease to divide. And so now they're no longer 00:33:04.27 capable of producing a rich growth of colonies on such a 00:33:09.29 plate. See, there's very little growth here. And we call that 00:33:13.27 senescence. So taking away telomerase is a bit of a 00:33:17.23 delay as the cells go through about 50 or so cell 00:33:20.06 generations, 50 to 80, and then the cells' telomeres get 00:33:24.05 too short, and they undergo senescence. We've 00:33:27.12 scrutinized more closely what happens to cells at this 00:33:31.09 point. Interestingly, a few cells do survive, and in fact, 00:33:42.09 those cells eventually become quite capable of producing 00:33:46.26 good growth on a plate, but they're different. These cells 00:33:51.05 now have very heterogeneous and long telomeres. I'll 00:33:55.01 show you these in a more graphical form in a moment. A 00:34:01.17 gene required for this to happen, for such "survivors," as 00:34:06.03 we call them, to appear, is the gene RAD52. For at least 00:34:13.05 one of these pathways, the RAD50 gene is also required. 00:34:17.11 What are RAD52 and RAD50? These are in the 00:34:22.07 homologous recombination pathway. They're needed for 00:34:27.02 recombination and therefore, to get survivors required 00:34:30.28 recombination. Similar phenomena have been seen in 00:34:35.16 mammalian cells. They've been less well characterized 00:34:38.16 genetically, but similar survivor cells have been seen, and 00:34:42.23 they're called ALT cells in mammals, for "alternative 00:34:47.18 lengthening of telomeres"... ALT cells. So, without 00:34:54.10 telomerase, most cells go through senescence, but rare 00:34:57.28 cells can survive. So, that is another way that 00:35:04.21 chromosomes can survive, but interestingly enough, ALT 00:35:10.04 is not normally seen in most normal cells. Now we 00:35:16.17 wondered why, because on the face of it, it looks as if 00:35:20.05 these cells are growing perfectly well. Although if you look 00:35:24.06 more closely, you will see that some of the cells are not 00:35:27.04 living well, but the great majority can survive with these 00:35:30.14 very long telomeres, that they can keep recombining, and 00:35:34.03 keep the population up that way. We asked what's 00:35:38.01 different. So first of all, we quantified very carefully the cell 00:35:43.19 growth, so this is just one way of showing it. The colony- 00:35:46.26 forming units, which is a measure of viability, as the cells 00:35:51.21 went through the progression of shortening telomeres, 00:35:54.17 getting to this point when they get to senescence, and 00:35:56.14 then those rare survivors now overgrew, and now you 00:36:00.29 can see them taking over and becoming the population. 00:36:03.23 So they're growing again quite well. And we looked at the 00:36:07.23 gene expression profile in such cells. First of all we looked 00:36:11.19 at the telomeres, just to make sure that the telomeres 00:36:14.20 were doing what I showed you diagrammatically before, 00:36:17.28 yes they are, so here's the telomeres. And this is what's 00:36:20.20 called a Southern blot, where we're probing with a 00:36:23.00 telomeric DNA sequence. And the most important are 00:36:26.13 these bands down here, that's the easiest to look at 00:36:28.21 initially, because this is a lot of the telomeres. You can 00:36:31.19 see they're of a certain length, and as they get shorter 00:36:34.11 they run faster and faster in the gel, so you can see 00:36:37.07 they're getting shorter and shorter with these successive 00:36:39.21 days of passaging of cell divisions without any telomerase 00:36:44.13 present. Now, there are very few cells here actually, but 00:36:49.09 we loaded enough DNA so you can see what little 00:36:52.11 telomeric DNA there is. And then the cells start growing 00:36:55.00 well again, and what you see is now the telomeres, the 00:36:57.29 pattern has changed, they're very heterogeneous, and 00:37:00.19 there's much longer telomeres, you can see they're longer 00:37:03.18 than these ones here. So that's the telomeric profile. Now, 00:37:13.20 let's look at the gene expression profile. This is what's 00:37:18.01 called a microarray experiment, and in this experiment, 00:37:22.27 one compares the pattern of gene expression by looking 00:37:26.07 at the messenger RNA levels for all of the genes in the 00:37:30.05 genome. And one asks, does a particular gene, 00:37:34.11 compared with a reference, which is right at the 00:37:36.17 beginning, when everything is growing well, does a 00:37:38.26 particular gene's level of expression become higher or 00:37:42.09 lower. If it becomes lower, it gets greener and greener. 00:37:46.12 And so each of these going across is a time point, each 00:37:57.23 line in the column represent a single gene, and each of 00:38:01.05 these horizontal areas represents the day at which the 00:38:08.13 RNA was taken out of the cells and analyzed in this way. 00:38:11.26 And so what we find, and it's all compressed and you just 00:38:14.23 need to look at the pattern, was that about 650 genes 00:38:19.07 changed their expression, and they got either lower levels 00:38:23.22 of expression (and we'll talk about this in more detail, and 00:38:27.20 that's shown in the green) or higher levels of expression 00:38:31.09 (and that shows up as red). Now right away what you can 00:38:35.15 see is that the maximum changes occurred right when the 00:38:38.12 cells were approaching and into senescence. Six- 00:38:43.23 hundred and fifty genes is something like 10% of the 00:38:48.09 genes in the genome, which is about 6000 to 7000 00:38:52.22 genes. So about 10% of the genome shows a change in 00:38:58.03 expression, particularly at senescence, but also, as I'll 00:39:03.05 show you, some genes remain changed even when the 00:39:08.03 cells are growing well, maintaining their telomeres through 00:39:11.26 this recombination mechanism. So one can analyze all the 00:39:17.02 patterns of genes whose expression has changed and 00:39:21.00 compare them with known patterns of gene expression 00:39:24.20 changes that have been looked at in a variety of 00:39:28.00 experimental settings by the many people in the yeast 00:39:33.05 genomics and gene expression community, who've 00:39:36.17 looked at gene expression changes. So one can 00:39:38.28 compare what we see here with what's been seen in 00:39:42.04 other experiments done by many other groups looking at 00:39:46.00 many other conditions of changes to the cells in yeast. So 00:39:53.19 what we found was that in fact, this pattern we observed 00:39:57.04 was unique, and we gave it a name: the telomerase 00:40:00.19 deletion response, the TDR. Now we found a set of 00:40:05.02 genes uniquely upregulated in response to the deletion of 00:40:09.17 telomerase RNA, and we called that the "telomerase 00:40:12.04 deletion signature." It was a particular group that 00:40:15.08 behaved as a group only when you deleted the 00:40:17.18 telomerase RNA, so that would be a group of those that 00:40:20.19 showed up as red, because they were upregulated. And 00:40:24.01 that hadn't been seen by any other manipulation. So the 00:40:27.16 cells indeed were sensing that they were losing 00:40:30.16 telomerase, and they responded in a particular way, by 00:40:34.08 changing their physiology, which we read out as a 00:40:36.21 change in gene expression profile. Right at the stage of 00:40:41.27 senescence, when I showed you that there were a lot of 00:40:44.13 genes that particularly were turned up or down in their 00:40:48.03 activity, there was a particular known DNA damage 00:40:54.01 response profile that was very prominent. That actually 00:40:57.25 makes a lot of sense. If you remember, I told you that, 00:41:00.24 when telomeres become short, they will become prone to 00:41:04.05 fusions, and now when they fuse, then chromosomes will 00:41:07.21 get ripped apart as they try to go through the ensuing cell 00:41:11.23 divisions, because the chromosomes with fused telomeres 00:41:14.26 will now have two centromeres that will pull apart in 00:41:17.15 mitosis, breaking the chromosomes at random positions, 00:41:21.09 and causing essentially genomic havoc, which can 00:41:23.27 quickly lead to cell death if the cells try to keep dividing. 00:41:28.09 And there's a damage response that takes place when 00:41:31.23 DNA breaks appear, and this is what we see, so this 00:41:34.29 makes very good sense. At senescence, we see a 00:41:37.14 damage response. What was very intriguing was that we 00:41:41.03 also saw others things. We saw what's called an 00:41:42.25 "environmental stress" cellular response. This is a 00:41:46.17 common set of genes that are upregulated and 00:41:49.00 downregulated in response to various insults, chemical 00:41:52.28 insults of various kinds, but it's a common set of genes, so 00:41:57.09 this is called the environmental stress response, and we 00:41:59.19 saw that response occurring, particularly at senescence. 00:42:05.05 And there was a change to an aerobic metabolism 00:42:08.15 program, and indeed we found that the number of 00:42:10.05 mitochondria at senescence went up enormously. Very 00:42:13.11 intriguing observations, not expected. Now, the survivors 00:42:20.02 were fascinating because, even though I showed you 00:42:22.26 they appear to grow quite well, they had a gene 00:42:26.15 expression transcriptional profile which was distinct from 00:42:29.24 the wild-type cells. And what persisted was a subset of 00:42:34.27 the environmental stress response genes that stayed on, 00:42:39.27 so what's interesting is that those cells, which appear to 00:42:42.10 be growing well on the surface, "stoics," they're really 00:42:45.27 hurting; their physiology is different, and they sense 00:42:49.25 themselves as being under what they sense as a cellular 00:42:53.23 stress. So, you can have cells that grow well without 00:42:58.20 telomerase, but if you look at the cells, these cells are 00:43:02.05 behaving as though under an environmental stress, and 00:43:05.25 so they indicate by their gene expression profiles a cellular 00:43:09.21 stress response. So in fact, cells do grow better with 00:43:15.04 telomerase than without it, even though superficially they 00:43:18.20 look similar, cells growing without telomerase maintaining 00:43:22.00 their telomeres via this recombination type of mechanism, 00:43:25.24 which is presumably somewhat more haphazard, is putting 00:43:28.27 a continuous stress on the cells, even though they've 00:43:32.04 adapted to it by a stress response. So, what we learned 00:43:38.09 then is that yeast lacking telomerase, using recombination 00:43:42.09 to maintain telomeres, do grow well, but they're under 00:43:45.10 continuing cellular stress, and this is what these gene 00:43:48.29 expression profile analyses showed us. So, let's go back 00:43:55.28 to telomerase and consider again what it's doing. If you 00:44:00.19 have plenty of telomerase, as indeed yeast cells normally 00:44:05.00 do, unless we genetically or in other ways ablate the 00:44:08.13 action of telomerase... if you have plenty of telomerase, 00:44:11.25 then there's kind of a homeostasis of the telomeres. The 00:44:15.21 telomeres stay within a certain range of lengths, and 00:44:19.06 remember I said at the very beginning of this lecture that 00:44:22.23 telomeric repeats are different in different molecules of a 00:44:27.02 population of cells, in different DNA molecules. And in 00:44:31.03 fact, they distribute around an upper and a lower limit. 00:44:35.27 And telomerase is one of the things that's crucial for 00:44:39.06 keeping them within this limit, and if you don't have 00:44:41.25 telomerase, then because of the DNA replication 00:44:46.05 problems, they gradually fall below. But they don't get too 00:44:49.25 long either, and a great many factors limit the action of 00:44:54.01 telomerase on telomeres as well, so they don't get too 00:44:57.05 long, and telomerase is also regulated on telomeric ends, 00:45:03.22 so that as the telomeres get shorter, then telomerase has 00:45:07.07 a higher probability of acting on the telomeres. This is a 00:45:09.25 complex system, we call it a homeostasis type of system, 00:45:14.27 and it balances out the lengthening and shortening 00:45:17.23 processes, so that the net result is that the telomeres stay 00:45:21.02 some average length within limits, and therefore the cells 00:45:24.21 keep dividing. Now, everything I've said implied that, if 00:45:32.22 you took away telomerase, there would be quite a delay 00:45:37.11 before any response was seen, and indeed, at a gross 00:45:42.09 level that is true. But if one scrutinizes the cells very 00:45:46.21 carefully, one sees that actually, things are a little bit 00:45:50.12 different. So an experiment in yeast was done, which was 00:45:54.26 to remove telomerase, as I showed you, and if you look at 00:45:58.00 the cells, I showed you growing on the plate, lots of 00:46:01.10 colonies, right away things look pretty okay. But if one 00:46:07.12 used very sensitive, quantitative molecular probes to look 00:46:12.05 at the telomeres in those cells, still with their long 00:46:16.22 telomeres, one found that there were occasional... every 00:46:20.22 few thousand cells or so had very short telomeres, and 00:46:24.28 they would actually fuse with a DNA break that was 00:46:28.13 induced to occur in those cells, something a telomere 00:46:33.05 never should do if it's a properly kept, functional telomere. 00:46:37.24 So, one in a few thousand cells had dysfunctional 00:46:42.17 telomeres, as manifested by the fact that they underwent 00:46:46.09 these normally forbidden fusion events. So immediately, 00:46:52.23 there was a response, even if one made the telomeres all 00:46:55.25 longer and then took away telomerase, one still saw this, 00:46:59.16 even when the bulk telomeres were long, one could see 00:47:02.16 this kind of aberrant dysfunctional telomeres immediately 00:47:09.03 after loss of telomerase. So this is one piece of evidence 00:47:14.20 that telomerase actually is protecting the ends of 00:47:18.11 telomeres, even when they're plenty long enough, 00:47:20.25 telomerase itself seems to protect and sit at the ends of 00:47:24.21 telomeres, is one interpretation, and protects them from 00:47:29.14 catastrophic shortening and fusioning to a double-strand 00:47:32.10 break, even when telomeres are long. This doesn't 00:47:36.28 happen in a high number of the cells, but it happens 00:47:41.10 measurably, and so this protective function of telomerase 00:47:46.18 is important. So I've introduced you to telomerase, I've 00:47:52.15 shown you how it's important for the long-term growth of 00:47:54.21 cells because it is necessary for continuous replenishment 00:47:58.28 of telomeres, and I've introduced you to the first piece of 00:48:02.18 experimental evidence that telomerase has a protective 00:48:06.01 function in cells, even when telomeres are long. So in the 00:48:11.06 next lecture, we'll talk about telomerase and more of its 00:48:16.28 protective functions in different cellular settings. 00:48:22.26

Part 2: Telomeres and Telomerase in Human Stem Cells and in Cancer

00:00:07.08 Welcome to the second part of this three-part lecture 00:00:11.04 series that I'll be giving on telomeres and telomerase. In 00:00:14.21 part two, I'm going to discuss telomeres and telomerase in 00:00:18.13 human cells, and particularly I'm going to emphasize the 00:00:22.16 setting of cancer cells. Now you may recall from the first 00:00:27.04 lecture that the function of telomerase is to maintain the 00:00:31.23 telomeres and prevent them from shortening as cells 00:00:36.04 divide, because telomere shortening would otherwise 00:00:39.00 occur in the absence of telomerase, to compensate for 00:00:42.11 the shortening processes. And so, maintaining telomeres 00:00:46.14 allows the cells to keep on dividing. In human cells, we 00:00:54.09 can also see though that telomerase itself is protecting 00:00:58.22 telomeres, and so I want to show you one kind of 00:01:01.04 experiment for that. And the conclusion is going to be that 00:01:08.01 it's not just the bulk telomere length that matters, but the 00:01:11.18 presence of telomerase can determine whether a 00:01:15.02 telomere is seen by the cell as sufficiently long or not. The 00:01:21.26 experiment I'll show you was done in cultured human 00:01:25.09 cells. These were not cancer cells, and these were cells 00:01:28.22 that have normally extremely, extremely low levels of 00:01:31.22 telomerase, effectively, for our purposes, essentially no 00:01:35.16 measurable telomerase activity, and certainly their 00:01:37.25 telomeres are not maintained. Now I've shown you in very 00:01:40.26 simple diagrammatic form human telomerase, so this 00:01:44.20 would be the template sequence of the human 00:01:47.21 telomerase RNA, and this is the overhanging G-rich 00:01:53.17 strand. It's actually usually longer than this, but I've just 00:01:56.26 shown it as a simple diagrammatic form here. And this of 00:02:00.23 course is the duplex telomeric DNA that will be consisting 00:02:06.06 of hundreds or thousands of telomeric repeats, as you go 00:02:09.02 in toward the chromosome interior. And just as I showed 00:02:14.00 you for Tetrahymena telomerase, the template region is 00:02:17.28 copied, and so the DNA... for example, a DNA with three 00:02:22.15 Gs, two Ts, and an A, would sit down here on the 00:02:25.13 template, and then nucleotides would added, extending 00:02:28.13 along the template, thereby lengthening the DNA in this 00:02:34.01 reverse transcriptase reaction that telomerase carries out. 00:02:39.27 So what was done was to compare cells in which 00:02:43.29 telomerase was being expressed or not being expressed, 00:02:49.15 and look at the growth of the cells and the telomere 00:02:52.21 length. Now the protein TERT is the core protein that has 00:02:57.11 this enzymatic activity: the reverse transcriptase activity, 00:03:01.28 and probably other activities, as I will allude to. And so, in 00:03:06.29 these experiments, a particular mutation was made on the 00:03:11.07 TERT protein, it happened to be a very small change of a 00:03:13.27 few amino acids added to the very C-terminus, and what 00:03:18.29 this does is it doesn't affect the enzymatic activity, but it 00:03:22.25 does affect the ability of this enzyme to elongate 00:03:27.10 telomeres in cells, and it's called a hypomorph because 00:03:32.21 that refers to the fact that it has an insufficient function, 00:03:37.26 but it showed something useful. Now here's the 00:03:40.28 experiment, and I'm going to walk you through this rather 00:03:43.28 complex-looking slide. First of all, I want to show you a 00:03:47.14 growth curve of human fibroblasts in culture. What 00:03:51.10 normally happens is, if we look at the... this is cumulative 00:03:55.03 dilutions, which is just an operational term telling you how 00:03:57.29 many cell divisions are going on, and this is the number of 00:04:00.29 days. So if you culture human fibroblasts, normally what 00:04:04.08 you find is that the cells will continue to multiply for a 00:04:08.04 while, and then they'll cease multiplying any further, so the 00:04:11.19 curve flattens out, so you see they've undergone 00:04:14.11 something like 50 or so divisions. And if you put in a 00:04:19.02 control vector, which would be important, you get the 00:04:22.07 same curve. But if you put in the test vector, which 00:04:26.09 actually is expressing this form of the human telomerase 00:04:31.07 core protein TERT, because the fibroblast cells naturally 00:04:35.29 have enough telomerase RNA and the other components 00:04:38.14 of telomerase, all they're missing is the TERT, you just 00:04:40.27 have to add this in, and now you can restore telomerase 00:04:44.08 activity. But what's very interesting is the telomeres and 00:04:47.26 the cell growth. What you can see is, first of all, the cell 00:04:51.19 growth has been greatly extended. We've now made 00:04:54.15 these cells very much elongated in their lifespan 00:04:59.03 compared with the controls or the parental cells. Now let's 00:05:03.11 look at the telomeres. If we look at the telomeres in these 00:05:07.27 cells, the controls, we find that they're gradually becoming 00:05:12.07 somewhat shorter, and at this point here, they've pretty 00:05:15.13 much ceased to divide, so we're out here, they pretty 00:05:18.20 much cease dividing. So the telomeres on average are 00:05:21.24 about this long, and the cells have picked up the signal, 00:05:24.27 they've said the telomere length is shorter here than here, 00:05:28.01 and they've picked up the signal, and they've said we're 00:05:29.16 not going to divide any further. Now we've added the 00:05:32.27 hTERT. Now actually what happened was that, I told you 00:05:35.27 the cells continue to grow a long, long time. The 00:05:39.10 telomeres shorten, and then they steady out at some 00:05:43.11 much shorter length, so through here and through here, 00:05:46.09 they're actually growing quite well, but they're going with 00:05:48.19 much shorter telomeres. So in other words, these cells 00:05:55.13 can keep dividing for a long time with very short 00:05:58.09 telomeres, and the difference was they had telomerase 00:06:02.03 being expressed. So we used a trick just to separate out 00:06:06.05 the telomere lengthening property of telomerase from its 00:06:10.02 ability to stabilize telomeres, and we saw that in fact if you 00:06:13.27 have this telomerase present throughout, even though 00:06:17.04 the telomeres maintain short, they are perfectly stable, 00:06:21.09 and the cells can keep dividing. So this is the second 00:06:25.24 piece of evidence, I showed you the first piece of 00:06:29.00 evidence for you in yeast systems in the first part of this 00:06:33.05 lecture series, and now this is the second piece of 00:06:35.20 evidence that telomerase is having a protective function, 00:06:39.10 in this case, it's in human cells, and it's stabilizing 00:06:42.12 telomeres that otherwise would've been too short in its 00:06:45.08 absence. This is not unique to human cells, it's been seen 00:06:48.27 in yeast systems as well experimentally. So I just talked to 00:06:54.02 you about normal cells, and I told you that, in those 00:06:58.11 human fibroblasts grown in culture, there's very little 00:07:01.22 telomerase. So, now let's talk about where do we normally 00:07:10.09 find telomerase in human cells? Well, if you look in cells, 00:07:20.14 you find that it actually is on at times when the cells are 00:07:23.18 greatly proliferating during fetal development, and it does 00:07:26.25 remain active in certain proliferative cells. It's also found 00:07:31.15 active in stem cells, in cells that are activated to 00:07:35.24 proliferate, such as lymphocytes proliferating under the 00:07:41.20 response to, for example, a pathogen. One finds stem 00:07:47.01 cells, for example, in hair follicles; those have telomerase. 00:07:51.07 So one does find telomerase in cells that are stem cells 00:07:54.27 and various sorts of cells that are induced to proliferate. 00:08:02.17 And in fact in most other cells, one can find telomerase, 00:08:06.23 but it's in very low levels. So initially people thought there 00:08:10.03 was no telomerase in normal epithelial cells or fibroblasts 00:08:13.14 or endothelial cells, but closer scrutiny showed that in fact 00:08:17.20 there were real, definitely low and very downregulated, 00:08:21.16 but real levels of telomerase. And in the third part of this 00:08:29.02 three-lecture series, I will talk more about the telomerase 00:08:34.10 in the normal cells of people and tell you about some in 00:08:40.05 vivo studies that have been done. Now, cancer cells. 00:08:45.24 Cancer cells are infamous for their ability to keep on 00:08:50.11 multiplying. Now, they can do this for a variety of different 00:08:54.21 reasons. They lose their system of checks and balances 00:08:58.11 that prevent them from overmultiplying, and that's 00:09:01.07 because of a lot of genetic and epigenetic changes that 00:09:04.11 have taken place in their progression to become a 00:09:08.08 malignant cancer cell. Now, they are immortal cells, and 00:09:13.17 indeed, as you might expect, telomerase is on in these 00:09:16.19 cells, and in fact it's very high in the vast majority of 00:09:20.19 human cancers, particularly as they've got to the invasive 00:09:23.24 stages. And that makes a lot of sense based on what I 00:09:28.16 just told you, because if the cells are to keep on 00:09:31.01 multiplying, then they have to keep on replenishing their 00:09:34.01 telomeres. Now I'll you some more recent results in the 00:09:38.27 last few years, which also suggest telomerase may be 00:09:42.16 doing other things in human cancer cells. It is certainly 00:09:46.14 maintaining the telomeres, and that is important, but there 00:09:49.28 may be other functions as well. So I'll tell you some newer 00:09:52.21 findings about that. So, let's just recapitulate what I said 00:09:58.06 about where we find telomerase in humans. So it keeps 00:10:01.19 telomeres elongating and replenished, and therefore cells 00:10:04.26 can keep dividing in stem cells and, of course, I didn't 00:10:10.00 mention germ cells. Now of course we wouldn't be here if 00:10:13.05 we didn't have maintenance of telomeres from generation 00:10:16.03 to generation. Telomerase indeed is active in germ cells, 00:10:20.16 as well as stem cells of various kinds. As I said, it's 00:10:23.25 detectable in many normal cell types, and highly active in 00:10:27.03 the great majority of human cancers. And by the way, in 00:10:32.14 some of those human cancers in which you don't find the 00:10:35.09 high telomerase, one actually often finds this ALT 00:10:39.03 mechanism, but it's only a particular subset of cancers in 00:10:43.19 which one finds ALT being a prominent means of 00:10:47.25 maintaining telomeres. The great majority of the common 00:10:50.26 human tumors have highly active telomerase. So, 00:10:56.15 telomerase is highly active in human tumors. So one could 00:11:02.24 imagine inhibiting telomerase, and this might be a good 00:11:07.18 target for trying to inhibit the growth of cancer cells, if you 00:11:12.16 could inhibit telomerase in cancer cells. And so in 00:11:17.01 investigating this, some interesting things emerged. So 00:11:21.25 let's just think about what is expected to happen if you 00:11:24.09 don't have telomerase in the cancer cells. Well, as you 00:11:27.27 know, cells multiply, and their telomeres will progressively 00:11:31.05 become shorter and shorter if there isn't telomerase to 00:11:33.27 counteract that shortening, and so eventually when the 00:11:37.18 telomeres get too short, the cells would eventually cease 00:11:40.14 to divide, and the cells respond to those short telomeres 00:11:45.25 by either what's call the "senescence response," in which 00:11:49.02 the cells simply won't replicate their DNA anymore, or a 00:11:53.14 cell death response that can include an apoptotic 00:11:56.25 response, which involves an active cell suicide program 00:12:01.04 that can be induced by dysfunctional telomeres. Whether 00:12:05.15 it's senescence or cell death does depend upon the cell 00:12:09.11 type. So, the simple prediction, diagrammatically, would 00:12:14.03 be, if you didn't have telomerase, the cells now would 00:12:18.11 have shorter and shorter telomeres, and after some 00:12:20.29 number of divisions, eventually there would be cell death. 00:12:27.18 Without telomerase, it's been observed experimentally, 00:12:30.06 typically human cells lose their telomeric DNA at this kind 00:12:34.08 of rate. I've put 150-200 base pairs per cell division; that's 00:12:39.02 seen in a number of cultured cells and also in some 00:12:45.00 cancer cells in which telomerase has been inhibited. 00:12:48.21 Now, I told you that human telomeres are typically made 00:12:53.13 of hundreds, even thousands, of copies of telomeric 00:12:59.26 repeats, they're thousands of base pairs long, so at this 00:13:02.27 rate, it's going to take quite a lot of cell divisions before 00:13:06.03 the telomere gets short enough that the cells will have this 00:13:09.25 response. So that in fact is the prediction, and if you 00:13:15.19 inhibit the telomerase enzyme using, for example, a small 00:13:19.17 molecule inhibitor that inhibits the catalytic function of 00:13:24.00 telomerase that prevents it from carrying out that DNA 00:13:28.06 polymerase reaction by the reverse transcriptase 00:13:31.22 mechanism, if you inhibit telomerase in such a way, 00:13:35.22 indeed this is the observation. There's a gradual 00:13:38.03 shortening of telomeres, and eventually the cells cease to 00:13:41.01 divide. So this is seen in cancer cells in culture treated 00:13:44.20 with such an inhibitor. Now I've put a noted point up here: 00:13:50.29 but you're still keeping the telomerase ribonucleoprotein 00:13:55.15 (RNP) level high. You're not depleting the cells of the 00:14:00.16 enzyme, you are simply rendering that enzyme inactive. 00:14:07.25 And I make that distinction because of the next results I 00:14:10.29 want to tell you about. So, what was observed was quite 00:14:17.17 surprising. If one depleted telomerase, and the particular 00:14:22.04 way to knock the telomerase RNA down, then one found 00:14:26.07 there was a very rapid effect on human cancer cells. One 00:14:30.22 didn't see the long delay ensuing before the effect was 00:14:37.03 observed. So, now I'll tell you about those experiments. 00:14:42.29 So, reminding you again, here's human telomerase, it's 00:14:45.12 copying its RNA template, and of course the enzyme has 00:14:50.14 the TERT protein and the telomerase RNA, and it's the 00:14:54.10 RNA that is going to be depleted in these experiments. 00:14:59.15 How's the RNA depleted? A now commonly used 00:15:02.28 technique, which is called a knockdown using RNA 00:15:06.24 interference. Now the particular technique was to express 00:15:11.22 from a lentiviral vector, which you can introduce efficiently 00:15:16.03 into cells, a particular sequence which forms a double- 00:15:21.10 stranded RNA, and I've just shown the corresponding 00:15:23.27 DNA sequence here, and that double-stranded RNA can 00:15:27.23 interact with a cellular RNA that matches its sequence, 00:15:32.08 and eventually cause the breakdown of that RNA 00:15:37.00 through a complicated process known as RNA 00:15:39.19 interference. And so, siRNA refers to "short interfering 00:15:44.24 RNA," because such a short interfering RNA, which 00:15:49.00 involves introducing two strands of RNA complementary 00:15:53.24 to the target RNA, that is what causes the breakdown to 00:15:58.05 occur. Now when we did this experiment, so here's a 00:16:01.22 control where we're looking at a reference amount, here's 00:16:04.18 the telomerase RNA, one found that in fact the method of 00:16:09.16 introducing such a short interfering RNA by this kind of 00:16:14.06 construct here (the details don't matter), one could in fact 00:16:18.02 quite efficiently knock down the telomerase RNA in 00:16:22.17 human cancer cells grown in culture, for example, in 00:16:25.26 these breast cancer cells grown in culture. And so one 00:16:30.15 could knock it down almost down to about 10-12% of the 00:16:36.16 original level that one sees in the controls. So what 00:16:41.27 happens then? Now, if what I had told you was the case, 00:16:47.05 that we knock down the telomerase, then we would 00:16:49.20 expect to have to wait for a long time for the telomeres to 00:16:53.20 get short enough, if that were the only thing going on, 00:16:57.05 then we would have to wait before we saw any effect. 00:16:59.10 But right away, there was an effect on the growth of the 00:17:02.14 cells. Now just to put this into perspective here, I'll just 00:17:07.25 walk you through a couple of graphs. Here's the controls 00:17:10.00 of two kinds, and this is the number of cells here. And so 00:17:14.10 what you can see is that the cell number goes up and up 00:17:17.04 and up, as you might expect. The cells are dividing once 00:17:22.11 every day or two, and so you can see, after about four or 00:17:25.13 five days after the introduction of the construct that 00:17:29.29 knocked the telomerase RNA level down, that the cells 00:17:34.13 that received this are quite quickly growing more slowly 00:17:40.04 than the controls. So this must be occurring within a 00:17:43.07 couple of cell divisions. And here are some more controls 00:17:47.07 in which, here's the empty vector, here is the short 00:17:52.16 interfering RNA targeting telomerase RNA, and here's a 00:17:56.07 control version of that, in which it now no longer can 00:18:00.12 target the telomerase RNA, but everything else is the 00:18:03.08 same, and as you can see it behaves like the controls. 00:18:06.03 And so a great many control experiments were done to 00:18:09.29 show that this was a specific effect specific to knocking 00:18:13.27 down the telomerase RNA. This was done on bulk, 00:18:20.27 unselected cell populations. The cells were a melanoma 00:18:27.02 cell line that normally has very long telomeres. The bulk, 00:18:32.01 unselected populations is significant because what it 00:18:36.24 meant was that one could put the siRNA, the agent that 00:18:41.14 knocks the telomerase RNA down, into the cells at day 00:18:45.21 zero, and without even any selection, one could get 00:18:51.10 something like 80-90% of the cells that received this 00:18:54.15 construct, and in fact the ones that did grow out were 00:18:57.29 that low percentage that didn't receive the construct. So 00:19:01.21 in fact the effect is even stronger than what these curves 00:19:05.17 would indicate, because these ones that are growing out 00:19:09.08 are largely the ones that just didn't receive the construct. 00:19:17.07 Now, one of the things that is very important in human 00:19:21.16 cells to respond to DNA damage such as certain kinds of 00:19:26.09 telomeric DNA damage is the gene p53. Interestingly, 00:19:33.01 these effects did not require p53. Here is a pair of cell 00:19:37.19 lines that are otherwise isogenic. This is a colon cancer 00:19:41.17 cell line called HCT116, in these cells the p53 is wild-type, 00:19:46.26 here's the response to knocking down telomerase RNA. 00:19:50.26 In this otherwise isogenic cell line lacking p53 but 00:19:56.00 otherwise the same, this response is quantitatively the 00:20:00.00 same. So this is a different response from a classic DNA 00:20:05.14 damage response, and in fact we did not see DNA 00:20:08.20 damage response genes being induced here. We looked 00:20:14.03 at the telomeres by some molecular probing mechanisms 00:20:21.29 that allow one to see if the telomeres are uncapped, and 00:20:25.00 in fact they were not uncapped either. So we see, when 00:20:30.09 we knock telomerase RNA down, we rapidly see an 00:20:33.04 inhibition of cancer cell growth, these are cells that 00:20:36.06 normally have very high telomerase. p53 is not required 00:20:39.26 for this, suggesting it's not a classic DNA damage 00:20:42.18 response. And the telomeres, as far as we can see, are 00:20:46.01 not uncapped, and there's not DNA damage response. 00:20:50.01 Indeed, the telomeres hadn't had time to shorten 00:20:52.14 perceptively at all during this short timeframe. So the rapid 00:20:58.00 knockdown doesn't uncap the telomeres. That's not the 00:21:01.06 cause of the growth inhibition of these human cancer 00:21:04.25 cells. But interesting things happen very quickly to these 00:21:10.20 cells when you knock down telomerase RNA. I'm going to 00:21:15.11 show you this one experiment in which, in this experiment, 00:21:22.01 melanoma cells were grown in culture, and the RNA level 00:21:25.10 was knocked down by a completely different mechanism, 00:21:28.21 it's called a "ribozyme," and the purpose of it is to cleave 00:21:36.09 the telomerase RNA, causing it to break down, and so it 00:21:39.22 has exactly the same effect that I showed you for the 00:21:43.03 RNA interference: you knock down the telomerase RNA 00:21:46.10 level. A very interesting thing was seen. These are cells 00:21:51.14 that are growing in culture, and these are three flasks of 00:21:54.10 cells that either are control cells that just got the empty 00:21:57.08 vector. The cells are all growing on the bottom of the 00:21:59.11 flask, you can't see it. We're just looking at the medium, 00:22:03.02 the broth, the liquid medium in which the cells are 00:22:05.22 growing, and it's a nice, pinkish color here in the controls. 00:22:09.26 But here, in two separate versions of the cell lines that 00:22:15.26 received the construct that knocked the telomerase RNA 00:22:19.07 levels down, you can see that the medium has turned a 00:22:22.20 dark color. These were melanoma cells, so it was of 00:22:27.06 course very easy to wonder if indeed these cells were 00:22:31.17 now producing the pigment melanin in high amounts in 00:22:35.15 this and this but not in the control cells. And indeed, 00:22:40.13 analyses showed that that was exactly what was 00:22:42.22 happening. And in fact, a lot of interesting things 00:22:46.11 happened. These cells became more like their normal 00:22:50.21 counterpart cells. It was as though they were more 00:22:53.09 differentiated; they looked more dendritic in form, as 00:22:57.15 though they had become less cancerous. And indeed 00:23:00.18 their gene expression profiles had changed. So, knocking 00:23:06.01 down telomerase RNA also made these cells less 00:23:11.23 invasive. The more differentiated the cancers are, very 00:23:15.17 typically, the less invasive they are, and in fact, in 00:23:20.21 preclinical mouse model systems used, in fact metastasis 00:23:24.13 was knocked down. And that was seen whether one 00:23:27.15 knocked telomerase RNA down with the ribozyme, or with 00:23:31.13 the RNA interference mechanism, but in melanoma 00:23:36.17 models for cancer, using the experimental mouse system 00:23:41.01 in the laboratory, it was found that the metastasis is 00:23:48.10 decreased when the telomerase RNA is depleted. So 00:23:51.14 knocking telomerase RNA levels down changed the 00:23:55.01 nature of the cells, and it changed the nature of the cells 00:23:58.12 well before the telomeres became uncapped. So, as I 00:24:05.15 said, when you have plenty of telomerase, you expect 00:24:09.11 cells to be able to multiple indefinitely, and you don't 00:24:12.24 expect any effects to occur, of removing telomerase, until 00:24:18.28 a long time has elapsed. So, what we see by this abrupt 00:24:26.14 knockdown of telomerase level, by knocking down the 00:24:29.14 telomerase RNA, is we see rapid inhibition of cell growth, 00:24:33.04 p53 was not required, there was no DNA damage 00:24:37.11 response or telomere uncapping, and in fact metastasis is 00:24:41.11 reduced. Metastasis is reduced, what is going on? I briefly 00:24:50.29 mentioned, we looked at the gene expression profiles in 00:24:54.19 these cells, and we found that in fact that rapid 00:25:00.00 knockdown of telomerase RNA and that rapid slowing of 00:25:05.00 the cell growth is accompanied by and presumably 00:25:08.15 caused by cell cycle and tumor progression genes being 00:25:13.00 downregulated. Glucose metabolism is downregulated; 00:25:18.13 cancer cells typically have high rates of glucose 00:25:21.25 metabolism. That becomes downregulated in cells that 00:25:25.12 have less telomerase RNA. And the cells appear more 00:25:30.04 differentiated, making one wonder if there's a cell 00:25:33.06 differentiation program that's induced in these cells. 00:25:38.12 Unexpected changes, many open questions remain, but 00:25:43.00 these kinds of experiments have led to these 00:25:45.21 observations here that have two kinds of implications: 00:25:50.28 One is the scientific implication that telomerase has other 00:25:55.01 functions that are not solely mediated through its adding 00:25:59.27 telomeric DNA to the ends of the chromosomes, and the 00:26:03.25 other implication is that this makes telomerase an 00:26:08.16 interesting target for potential anticancer therapies. And 00:26:19.13 the take-home message that I think is most impressive 00:26:23.09 and unexpected was, there seems to be good reason to 00:26:27.15 think that high telomerase levels are promoting an 00:26:30.13 undifferentiated, "stem cell-like" phenotype, a very 00:26:34.16 unexpected observation. And in fact, now in other 00:26:38.09 systems, I won't have time in this lecture to go through it, 00:26:42.02 but there's now evidence that this is really the case, in 00:26:47.28 even noncancerous cells in certain model organisms 00:26:52.28 where this has been studied. 00:26:56.02

Part 3: Stress, Telomeres and Telomerase in Humans

00:00:03.18 Welcome to part three of the series of three lectures on 00:00:07.29 telomeres and telomerase. This third part is going to be on 00:00:12.01 stress, which I will define, and telomeres and telomerase 00:00:18.01 in humans. In the previous two lectures, you'll remember 00:00:23.00 that we talked about the fact that, if you have plenty of 00:00:25.20 telomerase, then homeostasis in the sense of telomere 00:00:29.24 length maintenance can be maintained. There's a 00:00:32.24 balance of lengthening and shortening processes on 00:00:36.11 telomeres, so that an average length of telomeres is 00:00:39.24 maintained, and the cells can keep dividing, if there's 00:00:42.20 enough telomerase and other conditions are met, 00:00:45.04 essentially indefinitely. So now I want to put this kind of 00:00:52.14 cellular and molecular information that we've been gaining 00:00:57.10 about telomeres and telomerase into perspective with 00:01:00.24 respect to humans and a question that is greatly of 00:01:05.24 interest to many people, and that is the question of, how 00:01:08.13 do we age? What underlies this familiar kind of 00:01:15.01 progression shown in this series of pictures here? The first 00:01:19.20 thing I'd like to emphasize is that aging is a multifaceted 00:01:23.29 process. I think there's plenty of reason to think that that's 00:01:28.19 the case, and there won't perhaps be a single answer. So 00:01:32.29 I will really focus on one particular aspect of it. And even 00:01:38.08 at the level of whole humans, we can think of multiple 00:01:42.28 facets of the process of aging, and one of them is the 00:01:47.00 observation that there's increased susceptibility to certain 00:01:50.11 kinds of diseases. And we can wonder about this, and 00:01:56.11 wonder how much of this is environmental (life factors, 00:02:00.14 stochastic), and how much of this is genetic? And of 00:02:05.05 course, no doubt there'll be much interaction between 00:02:09.12 those two. I'm going to talk about some work studying 00:02:15.19 telomere maintenance in humans and show an interesting 00:02:23.00 set of findings that suggest that the environmental and life 00:02:27.16 factors aspects can certainly play a role. And that is not to 00:02:33.05 say genetics is not important, and I'll show you a sort of 00:02:36.07 situation where it's clear genetics is important as well. So 00:02:42.20 if we think about this susceptibility to diseases and 00:02:46.10 mortality, one can wonder about the genetic and the 00:02:50.27 nongenetic aspects, and I'm going to show you a graph 00:02:53.26 that came from Gavrilova and Gavrilov just to give you an 00:02:57.11 example of the fact that the genetic and the nongenetic 00:03:01.17 components of aging are not just monotonic across the 00:03:05.13 decades of human adulthood. So this was a study of over 00:03:09.29 5000 daughters, of well-to-do daughters of well-to-do 00:03:14.01 families in Europe, and which there was good 00:03:16.09 genealogical information as to the fathers' lifespan and 00:03:19.27 the mothers' lifespan and the daughters' lifespan. And so 00:03:23.03 with this database of over 5000 daughters with such 00:03:26.03 complete information, and all of whom were well-to-do, the 00:03:31.27 question was asked: Of the daughters who lived to be 30 00:03:34.08 years and older, how did their lifespan relate to either their 00:03:39.06 fathers' lifespan, which is shown here, as I'll explain, or 00:03:43.07 the mothers' lifespan, and I'll just tell you about that. But it 00:03:46.11 was very similar to what I've shown you here for the 00:03:48.07 pattern of lifespan. And the relationship was this: If you 00:03:51.21 looked at daughters who lived to be a certain age and 00:03:56.08 asked what their life expectancy was compared with the 00:04:00.13 rest of the daughters who were born in those same years, 00:04:04.02 and compared with how old their fathers grew to be 00:04:07.27 before they died, in other words, what was their fathers' 00:04:09.29 lifespan, for people who had fathers who lived to be 75 00:04:15.07 years and older, there was really quite a strong 00:04:18.10 relationship between the greater the father's lifespan was, 00:04:23.24 the greater the chance that the daughter would live 00:04:26.22 longer than expected. And so this function, which is 00:04:30.15 called a residual, basically increases with parental 00:04:34.28 lifespan, at least just for the father, 75 years and older, 00:04:40.21 whose life expectancy was 75 years and older, and that 00:04:46.09 was the relationship expected if genetics is an important 00:04:52.22 contributor to the daughters' lifespan. So if the father lived 00:04:58.22 longer than 75 years, then the daughter's lifespan was 00:05:04.16 related to it in a way that said genetics was important. But 00:05:08.25 what about all the fathers who didn't live to be 75, who 00:05:11.17 lived to be less? There, the relationship was very random, 00:05:19.05 so in other words, it made little difference whether your 00:05:23.16 father had a lifespan of 40, or your father had a lifespan of 00:05:26.03 70 years, to what your lifespan, the daughter's lifespan, 00:05:31.14 was. So in other words, mathematically, this function 00:05:38.03 around zero, that's expected if actually inheritance is 00:05:41.16 unimportant. Very similar plot was seen when it was the 00:05:46.15 maternal lifespan instead of the paternal lifespan that was 00:05:51.04 plotted out. So in other words, for this great majority of the 00:05:56.15 daughters, over 70% of the daughters fell into this 00:06:03.11 category, in fact things that are not genetic (we could call 00:06:08.18 them environmental or stochastic, life factors... 00:06:11.09 nongenetic factors) were clearly more overwhelming 00:06:15.07 quantitatively. So the genetic component was much more 00:06:20.12 important to fathers with a long lifespan than it was for the 00:06:24.25 fathers who had this wide range of somewhat shorter 00:06:30.02 lifespans. It just says that the relationships can be more 00:06:33.02 complicated. And so many summaries have been put 00:06:36.19 forward for this kind of observation, and I've added in 00:06:40.08 another one, and so that is elderly subjects demonstrating 00:06:43.11 exceptional longevity, for which I have just told you there 00:06:46.26 is certain kinds of genetic information that argues that this 00:06:51.28 is really a genetically based phenomenon, exceptional 00:06:56.18 longevity, such elderly subjects have generally been 00:07:00.23 spared the major age-related diseases, such as 00:07:03.20 cardiovascular disease, diabetes, and cancer. Now those 00:07:07.14 diseases are responsible for most deaths in the elderly, 00:07:11.27 and those are the ones for which one can find a lot of 00:07:14.10 nongenetic causes as well. So, cardiovascular disease, 00:07:22.23 diabetes, and cancer: Responsible for most deaths in the 00:07:26.24 elderly, and for which there's a significant nongenetic 00:07:31.11 component. So, how can one analyze this further, and 00:07:35.01 what's this got to do with telomeres? So that is what I'm 00:07:37.03 going to tell you about now, some relatively recent 00:07:40.01 research which connects these with telomere 00:07:43.14 maintenance now in humans. So to remind you, if 00:07:49.18 telomeres are replenished by continuous telomerase 00:07:53.15 action, then the cells can keep dividing. And in humans, 00:07:59.26 when one looks at the distribution of telomerase activity in 00:08:04.21 different cell types, one does find telomerase in stem cells 00:08:08.05 and germ cells, these are cells that are expected to be 00:08:11.20 essentially immortal cells. One does find it in human 00:08:18.24 tumors, but I'm not going to talk about that in this part of 00:08:23.08 this lecture series. But one finds telomerase at regulated 00:08:29.08 but real levels in a variety of other normal adult cell types 00:08:34.17 as well. So telomerase in the setting of normal cells is 00:08:39.08 found in stem cells, which are necessary for replenishing 00:08:43.28 tissues throughout the lifespan, such as immune system 00:08:48.22 stem cells, hair follicle stem cells are a one class, stem 00:08:53.13 cells in the gut, there are a lot of cells that replenish 00:08:57.01 tissues throughout life, and one finds telomerase in these. 00:09:01.10 But one also does find detectable levels of telomerase in 00:09:04.27 many, many normal adult nonstem cells as well. So, let's 00:09:12.12 just reiterate these expectations. If you have plenty of 00:09:15.19 telomerase, then homeostasis is going to be balanced. 00:09:18.02 Cells will keep dividing, so that would describe the 00:09:20.13 situation for stem cells, for example. Now, if you have just 00:09:26.00 a little bit of telomerase, then what's the situation going to 00:09:30.03 be like in that case? Well, if you had no telomerase at all, 00:09:35.16 we discussed in the first lecture what would happen. 00:09:38.18 There would be progressive shortening of the telomeric 00:09:41.10 DNA, and eventually when the telomeres became too 00:09:45.21 short, there'd be cellular senescence, and that is indeed 00:09:48.17 observed in some cells that have very, very little 00:09:52.00 telomerase when they grow in culture. But, the situation in 00:09:57.02 normal cells in the human body, not cells growing in 00:10:00.09 culture but the cells freshly analyzed from humans, as 00:10:04.15 fresh as you can get cells, and you look at them directly, 00:10:09.16 it's a more intermediate situation. So for example, what's 00:10:15.07 the prediction? If you had some telomerase, right, you're 00:10:17.12 going to have the shortening processes going on, but 00:10:20.01 each time the cells are dividing, perhaps there's some 00:10:21.21 telomerase, and so it's trying to keep it up, but eventually 00:10:25.15 it loses the battle. So, if you had some telomerase, the 00:10:28.19 prediction is that, since you haven't completely 00:10:32.00 compensated for the shortening processes, while 00:10:34.24 telomerase might be able to keep up, eventually the net 00:10:38.09 shortening will overcome the elongation ability of 00:10:42.03 telomerase, and senescence will eventually ensue later 00:10:46.05 than if you had no telomerase. And if you had some, a 00:10:49.14 little bit less but still some telomerase, then you'd get 00:10:52.16 there, to the point of senescence, but somewhat faster 00:10:55.15 than those cells that had a bit more telomerase. And so 00:10:59.12 it's these situations of this sort of gray zone that seem to 00:11:03.02 be the situation in normal human cells. Now this is 00:11:10.12 important because normal human cells will include certain 00:11:15.08 kinds of stem cells that are required to keep replenishing 00:11:20.19 throughout life, and then the proliferating cells that arise 00:11:23.28 from those stem cells that are required to regenerate 00:11:26.22 tissues throughout life, such as the immune system. So 00:11:31.00 how do we age? So we've learned a lot as we have 00:11:35.08 studied telomeres and telomerase in the laboratory in 00:11:38.28 model organisms that can now start to feed into this 00:11:43.28 question of, in humans and in people susceptible to 00:11:49.15 diseases of aging, how does this happen, how do we 00:11:55.20 age? And the converse can also be true. And let me 00:12:03.13 show you this very striking example: the clinic, the 00:12:07.18 bedside in this little diagram here. The clinic can be very 00:12:12.21 informative as to what might be going on in terms of 00:12:16.04 underlying biology. And such was the case with 00:12:20.23 telomerase. There's a rare, inherited condition in humans, 00:12:26.19 it's very rare, but it's now been seen in some dozens in 00:12:29.19 families. When we have billions of people on the planet, 00:12:32.13 then even a very rare disease can sporadically show up 00:12:35.22 in amounts that cumulatively add up to dozens of families. 00:12:39.00 This has been seen for a particular disease, it happens to 00:12:41.20 have the name "dyskeratosis congenita," which you don't 00:12:44.12 really have to remember, because it's not the most 00:12:47.04 clinically relevant aspect that's important here. What's 00:12:51.00 important is that such individuals die from progressive 00:12:56.16 bone marrow failure, and they suffer a premature death, 00:12:59.29 they don't make it to old age. And what's the defect? The 00:13:03.25 defect is that a copy of the telomerase RNA gene, and 00:13:08.10 the RNA is a single-copy gene found in one copy on a 00:13:13.04 particular chromosome. You get one copy of the 00:13:15.12 chromosome from your mother, one from your father, or 00:13:17.15 vice versa, so you normally have two copies of the 00:13:20.08 telomerase RNA gene, indicated as this little, yellow bar, 00:13:24.11 and on these chromosomes. So these are the two 00:13:27.00 chromosomes that have the telomerase RNA gene, one 00:13:30.07 that you got from your mother and one that you got from 00:13:31.24 your father. When one of those copies don't work, then 00:13:37.12 the primary clinical problem is progressive bone marrow 00:13:40.03 failure. This, as I said, causes premature death. Indeed it 00:13:44.21 can cause it early adulthood, sometimes even childhood, 00:13:49.25 to middle age. Such individuals have telomeres that are 00:13:52.27 way shorter than the normal range seen in healthy 00:13:56.06 individuals who do not have this inherited condition, or in 00:13:59.15 their family members who do not inherit this mutation. The 00:14:04.23 characteristic is that the immune system has become 00:14:07.12 exhausted, it looks probably as though stem cells, or the 00:14:10.23 cells that arise from stem cells in the immune system, just 00:14:13.28 lose the ability to proliferate all the way through a normal 00:14:18.00 life expectancy. As I said, the progressive bone marrow 00:14:21.11 failure, which is the primary cause of much of the death, 00:14:26.01 that is the problem, and the immune system then cannot 00:14:30.14 keep being replenished, which normally happens of 00:14:33.22 course from cells in the bone marrow. Interestingly, such 00:14:36.23 individuals are cancer-prone, they have very short 00:14:38.29 telomeres, and as I said in the first part of this three- 00:14:45.19 lecture series, telomerase can act to protect the ends of 00:14:50.29 chromosomes, and if we have deficient telomerase, then 00:14:54.12 that may be one reason why the chromosome ends are 00:14:57.24 dysfunctional, they're not protected, and secondly, the 00:15:02.24 telomeres are simply shorter, so they're also more prone to 00:15:07.02 becoming dysfunctional. So the important result, which 00:15:12.01 was found in 2001 by Vulliamy et al., was that mutating 00:15:17.27 one or other copy, from mother or father (it didn't make a 00:15:20.08 difference who had passed the gene down), but if one of 00:15:24.23 the copies of the telomerase RNA gene was mutated, as 00:15:29.17 is the case in the family members affected by this inherited 00:15:33.02 disease, even though the other gene copy is wild-type, 00:15:38.04 these are the ensuing consequences. The important 00:15:44.11 message is that to get through a healthy, full human 00:15:48.18 lifespan requires both the telomerase RNA alleles, that 00:15:54.07 means gene copies, to be functional. That means the 00:15:57.23 quantity of the gene product matters. So this is what the 00:16:01.15 rare, inherited disease told, that even a 50% gene 00:16:05.00 dosage, one functional gene instead of two functional 00:16:08.22 gene copies, even a 50% gene dosage is a big problem in 00:16:14.24 the long lifespan of humans. Even though one can get 00:16:19.20 often to early adulthood, one cannot keep going 00:16:23.20 throughout life. And the thing that's going wrong is the 00:16:28.08 ability of systems such as the immune system to keep 00:16:32.16 replenishing itself, so the cell proliferation ability is 00:16:36.25 diminished. But that's a rare disease. Is this relevant to 00:16:42.21 what happens in the vast majority of humans who do not 00:16:45.15 have this rare disease, informative as it is? But now that 00:16:50.11 gave us strong clue to look. And so, what's the situation 00:16:55.26 for humans who are not ostensibly carrying any known 00:16:59.22 genetic disease, what's the effect of common variations in 00:17:04.29 how their telomeres are maintained in humans? Variations 00:17:08.16 caused by, for example, nongenetic differences between 00:17:12.25 individuals? So, to put it in perspective, a very interesting 00:17:18.19 observation was made in 2003 by Cawthon and 00:17:22.04 colleagues, and they reported in a cohort of about 140 00:17:26.19 unrelated people aged 60 years and older, when they had 00:17:32.17 shorter telomeres in their white blood cells, which are the 00:17:36.26 cells that are easily analyzed, and a blood sample taken 00:17:40.16 from a healthy individual can be analyzed for its white 00:17:43.07 blood cell telomeres and the average telomere length in 00:17:46.16 those cells... it was found that such individuals with 00:17:49.27 shorter blood cell telomeres have higher mortality rates 00:17:53.10 than those in the same cohort of people with longer 00:17:57.23 telomeres in those white blood cells. And specifically, the 00:18:01.23 shorter telomeres were associated with a three-fold higher 00:18:04.09 mortality rate from heart disease, and an eight-fold higher 00:18:07.26 mortality rate from infectious disease, and that's 00:18:11.05 interesting, given what I just told you about the fact that a 00:18:13.29 50% gene dosage, compared with the normal 100% gene 00:18:19.01 dosage of telomerase RNA, in people with the rare 00:18:22.29 disease, dyskeratosis congenita, that that was enough to 00:18:29.02 make their bone marrow depleted, and so they couldn't 00:18:32.00 fight off infections, in fact they die of infections. So that's 00:18:36.11 a very interesting observation in light of the genetic 00:18:41.00 observations. And indeed, for all causes of mortality in this 00:18:47.03 elderly cohort of people who were followed for 17 years 00:18:51.09 subsequent to the time their telomeres were analyzed, 00:18:56.06 overall, from all causes, the people with the shorter 00:19:00.07 telomeres had higher mortality rates, in other words, 00:19:04.09 poorer survival, compared with those with the longer 00:19:07.09 telomeres. So the telomere length was measured from 00:19:12.07 samples that had been stored away in the freezer, and 00:19:17.16 then 17 years later, it was taken out and analyzed, and 00:19:21.09 these comparisons were made, that it was the 00:19:23.22 subsequent 17 years that were related to whether the 00:19:28.20 telomeres at the beginning of the 17-year period had been 00:19:31.25 shorter or longer. So this was, if you will, a prospective 00:19:37.19 kind of study. They shorter telomeres predicted higher 00:19:42.17 mortality rates. But what caused what? Were the 00:19:48.00 telomeres shorter then because they were already fighting 00:19:50.15 off some of these diseases, and perhaps there'd been 00:19:52.24 more turning over of cells, and telomeres had become 00:19:56.05 shorter already, because people were already prone to 00:20:00.07 these sorts of mortality causes for example? Or was it the 00:20:03.26 other way around? Were the telomeres shorter, and did 00:20:06.16 that make the people less able in the subsequent 17 00:20:11.21 years to avoid mortality from these causes, and indeed, all 00:20:15.23 causes? These results did not say what came first or what 00:20:21.19 caused what. So now I want to tell you about some more 00:20:25.21 recent experiments which start to give one kind of 00:20:29.14 indication of causality in this otherwise, we know, quite 00:20:34.14 complex question of what is causing susceptibility to the 00:20:40.02 diseases of aging. And this was a collaboration which 00:20:43.23 involved studying chronic life stress, and relating that to 00:20:47.11 cellular aging (which in this case, will be related to 00:20:50.26 telomere maintenance) and risks of cardiovascular 00:20:53.24 disease. And our group collaborated with the group of Dr. 00:20:56.29 Elissa Epel at the University of California, San Francisco's 00:21:00.26 psychiatry department, and a number of other colleagues 00:21:04.26 who are all listed here. So now I want to tell you about 00:21:08.26 those findings, which are now done with human beings in 00:21:14.13 vivo, participants in these clinical studies. So the study 00:21:20.01 design was to study chronic psychological stress, and so 00:21:24.09 62 healthy, premenopausal women, aged 20-50 in this 00:21:28.10 group, who were the biological mothers of either a healthy 00:21:31.27 child (the control mothers) or a chronically ill child (a group 00:21:38.04 of caregiving mothers), they were studied. And so first of 00:21:42.02 all they did a standardized questionnaire which has had a 00:21:45.11 good track record as being an assessment of perceived 00:21:49.07 stress, and then the other parameter measured that I'll first 00:21:54.07 talk about was the number of years that the caregiving 00:21:56.28 mothers had been caregiving mothers, that was analyzed, 00:22:00.08 and a number of others that I will allude to later. So first of 00:22:03.28 all, the study design was to assess the perceived stress 00:22:09.09 for the whole group, both the control and the caregiving 00:22:12.28 mothers, and then the number of years of the situation of 00:22:17.15 being in this chronically stressful situation, being the 00:22:23.15 caregiver of their child, who's chronically ill from a variety 00:22:27.05 of causes. So, three markers of cellular aging were 00:22:32.27 looked at: telomerase activity, telomere length, and also a 00:22:35.27 measure of cellular oxidative stress, which is a ratio of two 00:22:40.10 compounds, F2-isoprostanes and vitamin E, and that was 00:22:43.14 just taken as an assessment of the oxidative stress 00:22:48.13 situation physiologically in these individuals. We'll focus 00:22:52.23 on telomerase activity and telomere length. So, this was 00:22:57.02 the study design, as I just went through. And so the 00:23:03.18 questions were: Were the level of perceived stress across 00:23:06.18 both groups of mothers and the duration of caregiving in 00:23:10.10 the caregiver group... were those quantifiable parameters 00:23:14.27 related to markers of cell aging (and the three were 00:23:18.03 telomerase activity, telomere length, and cellular oxidative 00:23:22.10 stress)? Now, just to show you how we measured 00:23:25.08 telomerase activity, what we did was we measured 00:23:28.04 telomerase activity in the white blood cells of these 00:23:31.06 individuals. These are all healthy individuals, but they 00:23:34.11 gave blood samples. One could analyze telomerase 00:23:37.07 activity, and this is the telomerase activity gel, we used 00:23:40.08 three different concentrations always to make sure that 00:23:43.02 we're in the linear range, comparing it with internal 00:23:45.27 controls of various kinds, and a reference sample, which 00:23:49.11 is shown here, and then we quantified all these bands, 00:23:52.21 made sure we're in the linear range, and related it back to 00:23:55.24 the number of viable cells. So we could simply treat 00:24:00.11 telomerase activity in white blood cells as a quantitative 00:24:05.01 parameter, a continuously varying, quantitative parameter. 00:24:09.05 And so across the group, you find typically the lowest and 00:24:12.18 the highest, it's about 20-fold difference, and across the 00:24:15.25 group, there's a log normal distribution of telomerase 00:24:18.12 activities per cell in the different humans in the studies, 00:24:24.02 and we've looked at that in a number of studies and 00:24:26.05 found that same thing. Just to give you a little bit more 00:24:30.06 detail, the oxidative stress assessment came from looking 00:24:35.00 at the ratio of isoprostanes per milligram of creatinine 00:24:38.05 (that's to normalize), over vitamin E, that sort of net 00:24:42.28 oxidative stress assessment, if you will. And this kind of 00:24:47.05 ratio has been used as one kind of marker of oxidative 00:24:50.05 stress and antioxidant defenses that are current in that 00:24:54.23 individual. So what was interesting was that right away 00:24:58.21 one could see that, quantitatively, the worse the 00:25:02.17 perceived stress (the higher the score of perceived stress, 00:25:05.26 that is, the worse it was), the lower was the telomerase 00:25:09.05 length, the lower was the telomerase activity by this assay 00:25:13.16 I've told you about, and actually the worse, the higher 00:25:17.03 was the oxidative stress index. Right across the range, 00:25:20.14 across the entire sample that included the control mothers 00:25:25.12 and the caregiving mothers. Very interestingly, the number 00:25:29.07 of years of caregiving, and obviously this was now in the 00:25:32.07 caregiving group, similarly was related in just the same 00:25:36.29 way, so the number of years of caregiving was related 00:25:40.25 directly to how much shorter the telomeres got, how much 00:25:43.24 lower the telomerase was, and how much higher the 00:25:46.22 oxidative stress index was. So this, just to show you a 00:25:56.06 couple samples of data, here's the high stress quartile, 00:25:59.21 and this was corrected very carefully against all sorts of 00:26:03.00 other parameters in this very well-controlled group, and 00:26:06.10 these relationships didn't go away. Here for example are 00:26:08.21 the data compared for the high stress and the low stress 00:26:13.28 quartiles for example, and you can see here's the 00:26:17.00 average, and here's the range for the high stress quartile, 00:26:20.26 and there's less telomerase, about half the amount of 00:26:23.22 telomerase on average as there is in the low stress 00:26:26.22 quartile. And again, the error bars say that this was quite a 00:26:30.18 significant observation, taken together with the number of 00:26:34.26 individuals analyzed. So this was a striking relationship, as 00:26:40.11 was the relationship of telomere length. Now, I'm just 00:26:43.09 going to point to the error bars, they're very small here. 00:26:46.05 This is the high stress, this is the low stress quartile, you 00:26:49.24 can see these are very different. Here we've controlled 00:26:52.28 for age and body mass index for example. Controlling for a 00:26:56.13 lot of other factors, one still cannot make this difference in 00:27:00.13 the high stress versus the low stress go away, so we are 00:27:06.14 left with the conclusion that the strong variable here is the 00:27:12.06 stress level. Here's the oxidative stress now. The high 00:27:16.04 stress quartile, the low stress quartile, again, distinct. So, 00:27:21.18 stress perception and caregiving duration are linked to 00:27:25.19 cell aging markers, as assessed by telomerase, telomere 00:27:30.03 length, and oxidative stress. So the idea of cell aging is, 00:27:33.06 of course, the more the cell has gone through replicative 00:27:36.17 divisions, then the more aged it is in the sense of going 00:27:39.13 down towards critically short telomeres, then that would 00:27:43.24 be reflected in lower telomere length and potentially lower 00:27:46.24 telomerase. Oxidative stress is going to assess the 00:27:50.17 damage to the molecules in the cells, another measure of 00:27:54.11 their potential aging. Now what are the causal directions, 00:27:59.04 what are the mechanisms? I'll give you two short kinds of 00:28:03.20 indications. First of all, what about the causal direction? 00:28:07.11 So we're seeing these associations, what caused what? 00:28:11.20 Now the number of years of caregiving was a very 00:28:13.28 important thing to think about. So this was the plot of that. 00:28:19.05 This is the number of years of the caregiving situation, 00:28:24.27 and you can see it ranged from actually one year in some 00:28:28.18 of the mothers, some of the mothers had had four, some 00:28:30.29 six, eights, and some had all the way as long as 12. And 00:28:34.25 that was, as you see, quite strikingly related to the 00:28:38.23 shortness of the telomeres, so the ones who had fewer 00:28:41.14 years had longer telomeres on average than the ones 00:28:44.00 who had more years. A similar relationship held for the 00:28:47.10 telomerase activity levels. So this gave us an important 00:28:53.07 kind of indicator, that chronic stress is what's wearing 00:28:57.02 down the telomeres, because of this number of years of 00:29:00.08 caregiving, that objective, stressful situation, being 00:29:03.19 directly related. So, we have one arrowhead in this 00:29:07.26 otherwise no doubt very complex set of interactions, and 00:29:11.20 that is chronic stress (this is now years of chronic stress of 00:29:16.03 a particular kind characterized by the difficulties of 00:29:20.18 caregiving in an unpredictable and often uncontrollable 00:29:24.14 situation)... chronic stress is causing lower telomerase and 00:29:29.07 shorter telomeres, and one could predict that the lower 00:29:32.13 telomerase is in fact leading to the shorter telomeres. Now 00:29:38.24 we know from our biological studies that such effects in 00:29:43.09 normal cells will reduce the ability, eventually, of cells to 00:29:47.05 replenish themselves. So that's an important piece of the 00:29:52.00 picture, because for many model systems and biological 00:29:55.29 studies of cells, that relationship we know is the case. So 00:30:01.00 in whole humans we're finding this arrowhead is in that 00:30:05.15 direction in this particular setting of the chronic caregiving 00:30:09.17 situation. Now, what about mechanisms? In such 00:30:13.12 individuals, when their stress hormones were measured, 00:30:17.06 so these are stress hormones that are produced in 00:30:20.03 response to the brain dealing with this chronic stress 00:30:24.08 situation, cortisol, epinephrine, norepinephrine levels are 00:30:28.04 higher in highly stressed individuals, they're also higher in 00:30:33.03 the low telomerase half of people than in the high 00:30:36.10 telomerase half of people. In other words, you had worse 00:30:39.06 stress hormones if you had low telomerase than if you had 00:30:43.18 high telomerase. This is not implying causality, but the 00:30:47.05 relationship is there, and so it's going to be very 00:30:50.03 interesting to ask whether these stress hormones indeed 00:30:53.16 cause the lower telomerase that is observed in the white 00:30:57.07 blood cells of these individuals. So now we have a new 00:31:02.15 connection here, which we can think of in this way. We 00:31:05.04 think of a signal input, if you will, to the brain, now that is 00:31:08.19 the chronic stress. Now thereafter, things will get very 00:31:12.07 complex, we'll have all sorts of brain-body interactions, we 00:31:16.06 can think of them as signal integration and processing, 00:31:18.25 but a readout, a very quantitative readout, is telomerase is 00:31:21.13 lower, telomeres are shorter. The question is, does that 00:31:27.03 matter at all? Is there any impact on disease? Fortunately, 00:31:33.16 in this study, a great many other parameters were 00:31:36.29 measured. So let's think about cardiovascular disease. 00:31:41.07 What do we know about that? Well, a great deal is 00:31:44.10 known about the risk factors for cardiovascular disease. 00:31:48.26 For example, in one of the largest epidemiological studies 00:31:52.17 of risk factors that's been undertaken, 29,000 people in 00:31:57.02 52 different countries in six different continents, six major, 00:32:04.08 prominent factors for cardiovascular disease risk were 00:32:08.25 shown to be this list here, in this study published by Yusuf 00:32:11.23 et al. in 2004. And they're all the ones you expect: 00:32:15.19 smoking, poor lipid profile, high blood pressure, diabetes, 00:32:18.17 abdominal obesity, psychological stress. Psychological 00:32:21.21 stress. Ahh, now this was the same kind of psychological 00:32:26.06 stress that's the kind that had been measured in this study 00:32:29.17 I just told you about, the mothers and caregiving mothers. 00:32:32.28 So actually this was the same kind of measure, and I just 00:32:35.15 told you that relationship had been found, that is, lower 00:32:40.20 telomerase activity, higher oxidative index, shorter 00:32:44.06 telomeres, had been found associated with the higher 00:32:48.21 levels of psychological stress, and the longer the duration 00:32:52.24 of the psychological stress. So we had one, but actually 00:32:56.08 in these very well-designed studies, all of these had also 00:33:00.04 been measured, and so in fact, there were measures for 00:33:03.19 either the risk factor itself or, in one case, a surrogate for 00:33:07.02 it. One of the risk factors is diabetes, that's a known risk 00:33:11.16 factor for cardiovascular disease. All the individuals in this 00:33:15.08 study I told you about were all healthy, so by definition, 00:33:18.08 nobody had diabetes, but in fact, fasting insulin and 00:33:21.21 glucose were measured, and those are, if they're high, a 00:33:24.28 risk factor for diabetes. So all of these risk factors, or in 00:33:30.01 this case, a surrogate for the risk factor, were all in place 00:33:34.29 in this study. So the question was, well, since these three 00:33:39.19 had been associated with the level of psychological 00:33:44.10 stress and the duration of psychological stress, what 00:33:47.11 about these? Interestingly, it turned out that the one thing 00:33:52.15 that emerged for all of these was, in this study which has 00:33:57.24 been published, lower telomerase activity in the white 00:34:01.08 blood cells. So that went with smoking, it went with the 00:34:05.01 bad cholesterol/blood lipid profiles, it went with worse 00:34:10.13 cardiovascular activity profiles, with higher fasting 00:34:16.00 glucose, higher adiposity, and as we talked about, higher 00:34:19.06 and longer psychological stress. So the one parameter 00:34:23.10 that emerged in this particular study was lower telomerase 00:34:27.14 activity, now which, as we have talked about, is 00:34:31.22 predicted to lead to lower telomere length, but that didn't 00:34:35.21 emerge in this particular study, perhaps because the 00:34:38.29 individuals were relatively young. So, just to show you for 00:34:45.16 smoking, here is the range of telomerase activity per cell 00:34:51.27 in the white blood cells for the nonsmokers, you see it 00:34:54.07 follows a broad distribution, in fact, this is a log normal 00:34:57.07 distribution for the whole group. Here are the smokers. 00:35:01.09 They're all very, very low telomerase, in fact, the p-value, 00:35:04.22 the probability that this would be observed just by 00:35:07.18 chance, is very, very low, so telomerase was very low in 00:35:11.20 the smokers. There's no causality implied here, this is just 00:35:15.02 the association. Here's a very interesting observation for 00:35:20.13 heart function. So one can take all the telomerase activity 00:35:25.26 numbers for the entire group and simply divide them down 00:35:29.02 the middle, it's called a median split, into those who have 00:35:32.25 low telomerase and those who have high telomerase. 00:35:37.01 Now, some laboratory analyses of these individual 00:35:41.05 participants were done. People sat for half an hour or 00:35:47.15 longer, and then their resting heart rate was measured, 00:35:50.27 and so here's the value for the high telomerase half, and 00:35:53.25 here's the value for the low telomerase half. You can see 00:35:56.06 right away that the low telomerase half have higher 00:36:00.02 heartbeat rate, just even resting, then the lower 00:36:03.15 telomerase half. The pulse pressure, the blood pressure, 00:36:08.03 is higher for the low telomerase people than the high 00:36:12.21 telomerase people, so in other words, these are less 00:36:16.10 healthy. There's a parameter called "high frequency heart 00:36:19.23 rate variability," basically the variability is how well the 00:36:23.17 heart is adapting to changing needs, so when it's high, 00:36:26.29 that's good, it's adapting well. So you can see it's higher 00:36:30.12 in the people with high telomerase than it is with the low 00:36:32.15 telomerase group. Everybody's sitting resting. Then, an 00:36:37.15 interesting other thing was done in these individuals. They 00:36:44.07 were asked to undergo a series of laboratory 00:36:49.04 psychological stressors, in a very controlled setting. So 00:36:52.24 they are asked to anticipate giving a short videotaped 00:36:56.21 speech, to give the speech, and then to do some very 00:36:59.20 unpleasant mathematics in a highly controlled 00:37:02.26 environment in which individuals who are trained 00:37:06.07 personnel look at them stony-faced, and so, that normally 00:37:11.09 elicits a very uncomfortable response. Interestingly, how 00:37:15.29 people responded depended on whether they fell into the 00:37:20.04 high or low telomerase group. So I'll just show you the 00:37:23.10 curves, it's quite striking. So what you expect is, of 00:37:26.12 course, your heart rate will go up as you become more 00:37:30.23 nervous and uncomfortable with this deliberately 00:37:34.01 uncomfortable, rather unpleasant task. And you can see 00:37:36.29 that the low telomerase people, their heart rate went way 00:37:39.10 up, much more (it was already resting higher), went up 00:37:41.29 much more in response than the high telomerase people. 00:37:46.03 Similarly, their pulse pressure went much higher, and then 00:37:51.14 eventually came down somewhat, than the high 00:37:55.21 telomerase people. And the heart rate variability showed a 00:37:58.07 nice, healthy response in the high telomerase individuals, 00:38:03.06 but a much less healthy lack of ability to adapt in the low 00:38:08.10 telomerase people. So, simply splitting all of the 00:38:12.21 telomerase activity measurements into just two halves, the 00:38:16.21 high half and the low half, showed very different heart 00:38:20.16 functions. And in all cases, the difference was that the 00:38:25.10 low telomerase group always had the unhealthy response 00:38:28.24 that is predicted to lead to have more of a cardiovascular 00:38:32.17 disease risk. So what we've seen then, in summary, the 00:38:37.11 low telomerase alone, even in the absence of obvious 00:38:41.01 telomere shortening, which may well happen later but 00:38:43.16 hasn't happened in this group, is associated with six major 00:38:47.06 risk factors, including chronic psychological stress for 00:38:51.03 cardiovascular disease in people, and so that raises an 00:38:55.18 interesting question of, is telomerase status in normal cells 00:39:01.02 of people an indicator of their disease risk for, for 00:39:05.08 example, cardiovascular disease? Now indeed, telomere 00:39:09.23 shortening has been seen a lot as something that is being 00:39:15.11 associated with disease risks, and indeed disease is 00:39:18.23 incident now in more and more cohorts. So just to finish 00:39:24.27 the idea, the idea is that if you have lower telomerase, we 00:39:28.03 know that will be one of the things that will help drive 00:39:30.27 telomere length down, because telomere length 00:39:33.03 maintenance will be less able to be at the optimum level if 00:39:38.10 telomerase is down. So that, as we know, reduces the 00:39:43.20 ability of cells to replenish themselves. Now, that then 00:39:53.14 makes us step back and say, well, what about telomerase 00:39:58.12 and genetic components? I've just talked to you about a 00:40:02.03 clearly nongenetic component, years of stress induced by 00:40:07.18 a chronic caregiving situation. Here, let's consider known 00:40:13.05 genetic defects. Mouse telomerase has been knocked 00:40:17.03 out in model organisms, and I told you about the effects of 00:40:21.10 losing function of half the telomerase gene dosage in 00:40:25.23 humans. Again, telomerase activity goes down and 00:40:29.10 telomere length maintenance is going down, and in this 00:40:32.00 case, the cause is known to be less telomerase, and 00:40:36.18 telomere length does indeed go down. And indeed, one 00:40:39.16 does see reduced ability of cells to replenish themselves 00:40:43.16 in these situations where, genetically, telomerase has 00:40:47.29 been knocked down, so one knows for sure that that is 00:40:51.00 leading to these effects. Now, what about disease 00:40:56.21 impact? Indeed, the genetic defects do lead to diseases, 00:41:03.07 and in mouse models in which telomerase is knocked out, 00:41:06.26 there is indeed evidence for diseases of the kind that can 00:41:11.29 include propensity for cardiovascular disease. Now, 00:41:16.27 what's been seen clinically for many years is chronic 00:41:19.24 stress, which we can think of as that signal input, clearly 00:41:22.27 has disease impact. This has been seen in a great many 00:41:28.04 different studies. Now we've added a new question: Does 00:41:34.13 that occur via lower telomerase? And that is the open 00:41:38.07 question. So as I said, what we know is we have one 00:41:42.10 arrowhead here, we know chronic stress indeed can 00:41:45.19 cause less telomerase and worse telomere maintenance, 00:41:50.12 shorter telomeres, which we know from our biological 00:41:53.11 studies does in fact affect whether cells can replenish 00:41:59.20 themselves. We know from the genetic studies that that 00:42:03.13 can lead to disease impact, but the question is, is this the 00:42:11.14 case in the human studies I've described to you? Is this 00:42:17.26 really what's causing the disease impact, via the lower 00:42:22.12 telomerase? Or is it that the chronic stress actually has its 00:42:27.14 disease impact because of unrelated reasons? Or, more 00:42:31.09 likely, is it a combination of the two? So that is where 00:42:34.26 things are standing right now. Finally, just let me show you 00:42:39.26 a lot of data that have been accumulating in the last few 00:42:43.25 years, in which low telomere length is being more and 00:42:48.04 more linked to rather common diseases of aging, such as 00:42:52.20 cancer, cardiovascular disease, vascular dementia, 00:42:56.19 various degenerative conditions, diabetes, and in fact, 00:43:00.02 risk factors overall for chronic disease. And a great many 00:43:03.19 studies have linked shorter telomeres in white blood cells 00:43:08.21 to these rather common diseases of aging or risk factors 00:43:13.02 for such diseases. So in a great many cohorts, low 00:43:17.27 telomere length is certainly linked to these diseases. The 00:43:21.20 causality of course is not determined by any of these 00:43:24.28 studies, but we think that we may have a clue from our 00:43:30.06 finding that chronic stress will have effects on lowering 00:43:33.19 telomerase, which is expected to lead to lower telomere 00:43:37.00 length. So back to this general statement about genetic 00:43:42.23 and nongenetic aspects of aging in humans and disease 00:43:47.00 susceptibilities that lead to mortality in humans, the 00:43:51.05 general observation is that elderly subjects demonstrating 00:43:54.26 exceptional longevity have generally been spared these 00:43:59.05 major age-related diseases, such as cardiovascular 00:44:02.16 disease, diabetes, and cancer, which are responsible for 00:44:05.19 most deaths, and for which I've just shown you the kind 00:44:09.11 of evidence that is beginning to accumulate suggesting 00:44:13.28 that there are nongenetic (as well as, of course, some 00:44:17.06 genetic) components for these diseases. And so these 00:44:26.21 nongenetic as well as genetic causes are obviously very 00:44:31.12 interesting, since these major diseases are responsible for 00:44:35.06 most of the deaths in the elderly. So how do we age? The 00:44:41.16 question has come from what we've learned in the lab 00:44:45.00 about cells and molecules: telomerase, telomeres, how 00:44:49.29 their continued maintenance affects abilities of cells to 00:44:53.19 maintain themselves. In animal models, one sees that, if 00:44:57.09 it's compromised, it can lead to cell death and mortality of 00:45:02.05 the organism. That knowledge has been carried to the 00:45:06.00 understanding of some of the diseases of aging and 00:45:11.11 back, and so this continuous sort of cycle of knowledge 00:45:16.25 and understanding, I think is going to an important part of 00:45:20.15 how we address the question of how do we age. So, let 00:45:26.02 me summarize with a little metaphorical picture. The 00:45:29.01 summary is that it's emerging that shortening of telomeres 00:45:31.28 in human cells is associated with shortening of life. So if 00:45:36.06 we think of a chromosome and the telomeric structure at 00:45:40.02 its end, I like to think of this structure as being a beautiful, 00:45:43.04 elaborate tree, and what we're trying to understand are 00:45:48.10 the sorts of things that lead this beautiful tree to erode 00:45:51.27 down to a stump. Thank you. 00:45:57.22

Talk Overview

Telomerase, a specialized ribonucleprotein reverse transcriptase, is important for long-term eukaryotic cell proliferation and genomic stability, because it replenishes the DNA at telomeres. Thus, depending on cell type telomerase partially or completely (depending on cell type) counteracts the progressive shortening of telomeres that otherwise occurs. Telomerase is highly active in many human malignancies, and a potential target for anti-cancer approaches. Furthermore, recent collaborative studies have shown the relationship between accelerated telomere shortening and life stress and that low telomerase levels are associated with six prominent risk factors for cardiovascular disease.

Speaker Bio

Dr. Blackburn is a leader in the area of telomere and telomerase research. She discovered the molecular nature of telomeres-the ends of eukaryotic chromosomes that serve as protective caps essential for preserving the genetic information – and discovered the enzyme telomerase, which replenishes telomeres. Blackburn is currently a Professor in the Department of Biochemistry and… Continue Reading

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This material is based upon work supported by the National Science Foundation and the National Institute of General Medical Sciences under Grant No. MCB-1052331.

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